Process for accelerated capture of carbon dioxide

ABSTRACT

The present invention generally relates to the removal of carbon dioxide from a gas stream, particularly a flue gas, hydrogen gas from a reformer, natural gas, or gas from a cement kiln. Immobilized enzymes for use in carbon capture and other systems are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Non-Provisional patent application of U.S.Provisional Patent Application Ser. No. 61/147,462, filed Jan. 26, 2009,and U.S. Provisional Patent Application Ser. No. 61/101,052, filed Sep.29, 2008, the entirety of which are herein incorporated by reference.

FIELD OF THE INVENTION

The present invention generally relates to the removal of carbon dioxidefrom a gas stream, particularly a flue gas, hydrogen gas from areformer, natural gas, or gas from a cement kiln.

BACKGROUND OF THE INVENTION

Technologies are being developed for capturing carbon dioxide (CO₂) fromindustrial gas streams to reduce energy costs and the environmentalimpact of CO₂ in the atmosphere. Major sources of CO₂ output includepower plants, cement kilns, natural gas processing facilities, ammoniaplants, and hydrogen plants. The captured CO₂ may have multiple uses,including sequestration, enhanced oil recovery, or accelerated algaegrowth. In the cases of hydrogen, ammonia, and natural gas, removal ofCO₂ is necessary to increase the value of the gas stream.

Currently several alternate CO₂ capture technologies are in variousstages of commercial practice and development. These include chemicalabsorption with amines (particularly monoethanolamine—MEA), physicaladsorption, membrane separation, and cryogenic distillation. Inaddition, technologies such as oxycombustion and Integrated GasificationCombined Cycle, which remove the carbon or non-oxygen gas componentsprior to combustion, are being considered as ways to eliminate flue gasremoval. Chemical absorption with amines is currently the lowest costmethod of CO₂ removal for the majority of gas streams, particularly forthe clean-up of low levels of CO₂ in natural gas. MEA systems are morereactive, and therefore preferred, but the energy load to remove theabsorbed CO₂ from the MEA, at about 4 million BTU/tonne of CO₂ is veryhigh and can require up to about one-third of a power plant's boileroutput.

SUMMARY OF THE INVENTION

Among the various aspects of the invention is a system comprising animmobilized carbonic anhydrase for removing carbon dioxide from a gasstream, particularly, a flue gas, hydrogen gas from a reformer, naturalgas, or gas from a cement kiln.

Another aspect is a process for removing CO₂ from a CO₂-containing gas,the process comprising contacting an aqueous liquid with aCO₂-containing gas to promote diffusion of the CO₂ into the aqueousliquid; and contacting the CO₂ in the aqueous liquid with immobilizedcarbonic anhydrase entrapped in a polymeric immobilization material tocatalyze hydration of the CO₂ and form a treated liquid containinghydrogen ions and bicarbonate ions, wherein the polymeric immobilizationmaterial either (i) stabilizes the carbonic anhydrase or (ii) comprisesa micellar or inverted micellar material.

Yet another aspect is the process described above wherein the process isperformed in a reaction vessel which comprises a bottom portionincluding a gas inlet and a liquid outlet, a top portion including aliquid inlet and a gas outlet, and a middle portion containing aplurality of particles coated with immobilized carbonic anhydraseentrapped in a polymeric immobilization material wherein the polymericimmobilization material either (i) stabilizes the carbonic anhydrase or(ii) comprises a micellar or inverted micellar material. The processcomprises contacting an aqueous liquid which enters the liquid inlet andflows downward in the reaction vessel with a CO₂-containing gas whichenters the gas inlet and flows upward in the reaction vessel to promotediffusion of the CO₂ into the aqueous liquid and catalyze hydration ofthe CO₂ in the aqueous liquid in the presence of the immobilizedcarbonic anhydrase to form a treated liquid containing hydrogen ions andbicarbonate ions and a treated gas; and evacuating the treated liquidfrom the liquid outlet and evacuating the treated gas from the gasoutlet.

A further aspect of the invention is a reaction vessel for removing CO₂from a CO₂-containing gas comprising a bottom portion containing a gasinlet and a liquid outlet, a top portion containing a liquid inlet and agas outlet, and a middle portion containing a plurality of particlescoated with carbonic anhydrase entrapped in a polymeric immobilizationmaterial wherein the polymeric immobilization material either (i)stabilizes the carbonic anhydrase or (ii) comprises a micellar orinverted micellar material. The carbonic anhydrase is capable ofcatalyzing hydration of CO₂ into hydrogen ions and bicarbonate ions.

Yet another aspect is an enzyme immobilized by entrapment in a polymericimmobilization material, the material being permeable to a compoundsmaller than the enzyme and the enzyme being modified ionically orcovalently by a hydrophilic, hydrophobic, or amphiphilic moiety.

An enzyme immobilized by entrapment in a polymeric immobilizationmaterial, the immobilization material being permeable to a compoundsmaller than the enzyme and having the structure of either Formulae 5,6, 7, or 8:

wherein R₂₁ and R₂₂ are independently hydrogen, alkyl, or substitutedalkyl, provided that the average number of alkyl or substituted alkylgroups per repeat unit is at least 0.1; R₂₃ and R₂₄ are independentlyhydrogen, alkyl, or substituted alkyl, provided that the average numberof alkyl or substituted alkyl groups per repeat unit is at least 0.1;R₂₅ is hydrogen, alkyl, or substituted alkyl, provided that the averagenumber of alkyl or substituted alkyl groups per repeat unit is at least0.1; R₃₂ and R₃₃ are independently hydrogen, alkyl, aryl, or substitutedalkyl, provided that the average number of alkyl or substituted alkylgroups per repeat unit is at least 0.1 and m, n, o, and p are an integerof at least 10.

Another aspect is a system for removing CO₂ from a CO₂-containing gascomprising first and second reaction vessels, the first reaction vesselbeing the reaction vessel described above and the second reaction vesselcontaining particles coated with carbonic anhydrase entrapped in apolymeric immobilization material wherein the carbonic anhydrase iscapable of catalyzing conversion of the hydrogen ions and thebicarbonate ions into concentrated CO₂ and water.

A further aspect is a process for removing CO₂ from a CO₂-containing gascomprising contacting an aqueous liquid with a CO₂-containing gas topromote diffusion of the CO₂ into the aqueous liquid; and contacting theCO₂ in the aqueous liquid with immobilized carbonic anhydrase tocatalyze hydration of the CO₂ and form a treated liquid containinghydrogen ions and bicarbonate ions. The aqueous liquid comprisesmethylamine, ethylamine, propylamine, iso-propylamine, butylamine,iso-butylamine, sec-butylamine, tert-butylamine, pentylamine,iso-pentylamine, sec-pentylamine, tert-pentylamine, hexylamine,iso-hexylamine, sec-hexylamine, tert-hexylamine, ethylenediamine,(2-methylbutyl)amine, 2-aminopentane, 3-(tert-butoxy)propylamine,2-amino-6-methylheptane, 1-ethylpropylamine dimethylamine, diethylamine,dipropylamine, dibutylamine, dipentylamine, dihexylamine,N-ethylmethylamine, N-isopropylmethylamine, N-butylmethylamine,N-ethylisopropylamine, N-tert-butylmethylamine, N-ethylbutylamine,3-isopropoxypropylamine, chloro(diethylamino)dimethylsilane,2,2′-(ethylenedioxy)bis(ethylamine),1,3-bis(chloromethyl)-1,1,3,3-tetramethyldisilazane,N-tert-butylisopropylamine, N,N-diethyltrimethylsilylamine,di-sec-butylamine, trimethylamine, triethylamine, tripropylamine,tributylamine, dimethylpropylamine, diethylpropylamine,N,N-diisopropylmethylamine, N-ethyldiisopropylamine,N,N-dimethylethylamine, N,N-diethylbutylamine, 1,2-dimethylpropylamine,N,N-diethylmethylamine, N,N-dimethylisopropylamine,1,3-dimethylbutylamine, 3,3-dimethylbutylamine, N,N-dimethylbutylamine,or a combination thereof.

Yet another aspect is the process, reaction vessel, or system describedabove wherein the carbonic anhydrase is a bovine carbonic anhydrase or ahuman carbonic anhydrase. Particularly, the carbonic anhydrase is abovine carbonic anhydrase II, a human carbonic anhydrase IV, or amodified human carbonic anhydrase IV.

Other objects and features will be in part apparent and in part pointedout hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of pH versus time for a reaction of carbon dioxideconversion to carbonic acid for a reaction catalyzed by carbonicanhydrase and a reaction with no carbonic anhydrase catalysis whereinthe experiment was performed in 0.5M Na₂CO₃, 50 sccm CO₂, at 4° C., andusing 25 mg (0.5 mg/mL) carbonic anhydrase.

FIG. 2 is a schematic of a CO₂ absorber coupled with a CO₂ desorber.

FIG. 3A is a schematic of the packed bed reactor used to collect thedata presented in Example 1.

FIG. 3B is a schematic of a carbon capture system having an absorber anda stripper.

FIG. 4 is a graph of volume of 2M sodium hydroxide added to the reactorto keep the pH constant as the CO₂ is converted to bicarbonate versustime wherein the carbonic anhydrase was treated with Triton-X 100 andimmobilized in tetraethylammonium-modified Nafion®.

FIG. 5 is a graph of volume of 2M sodium hydroxide added to the reactorto keep the pH constant as the CO₂ is converted to bicarbonate versustime wherein there was no carbonic anhydrase in the reactor.

FIG. 6 is a graph of the pH versus time for a reaction of bicarbonateconversion to carbon dioxide catalyzed by non-immobilized carbonicanhydrase and no enzyme as described in example 2A wherein theexperiment was performed in 0.1M Na₂CO₃, 50 sccm CO₂, at 40° C., andusing 0.313 mg/mL carbonic anhydrase and a run time of 50 minutes.

FIG. 7 is a graph of the pH versus time for a reaction of bicarbonateconversion to carbon dioxide catalyzed by non-immobilized carbonicanhydrase and no enzyme as described in example 2A wherein theexperiment was performed in 0.1M Na₂CO₃, 50 sccm CO₂, at 40° C., andusing 0.313 mg/mL carbonic anhydrase and a run time of 16 hours.

FIG. 8 is a graph of the pH versus time for a reaction of bicarbonateconversion to carbon dioxide catalyzed by non-immobilized carbonicanhydrase as described in example 2B wherein the experiment wasperformed in 0.1M, 0.5M, and 2M Na₂CO₃, 50 sccm CO₂, at 40° C., andusing 0.313 mg/mL carbonic anhydrase.

FIG. 9 is a graph of the pH versus time for a reaction of bicarbonateconversion to carbon dioxide catalyzed by non-immobilized carbonicanhydrase as described in example 2C wherein the experiment wasperformed in 0.5M Na₂CO₃, 50 sccm CO₂, at 20° C. and 40° C., and using0.313 mg/mL carbonic anhydrase.

FIG. 10 is a graph of the pH versus time for a reaction of bicarbonateconversion to carbon dioxide catalyzed in two different runs byimmobilized carbonic anhydrase as described in example 2D wherein theexperiment was performed in 0.1M Na₂CO₃, 50 sccm CO₂, at 40° C., andusing 0.313 mg/mL carbonic anhydrase treated with Triton-X 100 andimmobilized in tetraethylammonium-modified Nafion®. An experimentwherein no carbonic anhydrase catalyst was used was also performed.

FIG. 11 is a picture of polysulfone beads and a cross sectional picturewith and without a soluble dye present.

FIG. 12 is a graph of pH versus time for a reaction of carbon dioxideconversion to carbonic acid catalyzed with carbonic anhydraseimmobilized in polysulfone, free carbonic anhydrase, and no enzyme asdescribed in example 1C wherein the experiment was performed in 0.5MNa₂CO₃, 50 sccm CO₂, at 4° C., and using 25 mg (0.5 mg/mL) carbonicanhydrase.

FIG. 13 is a graph of pH versus time for a reaction of carbon dioxideconversion to carbonic acid catalyzed with carbonic anhydraseimmobilized in alginate and coated with polysulfone, free carbonicanhydrase, and no enzyme as described in example 2A wherein theexperiment was performed in 0.5M Na₂CO₃, 50 sccm CO₂, at 4° C., andusing 25 mg (0.5 mg/mL) carbonic anhydrase.

FIG. 14 is a graph of pH versus time for a reaction of carbon dioxideconversion to carbonic acid catalyzed with carbonic anhydraseimmobilized in crosslinked poly(vinyl benzyl chloride) (PVBC) and noenzyme as described in example 3 wherein the experiment was performed in0.5M Na₂CO₃, 50 sccm CO₂, at 4° C., and using 80 mg carbonic anhydrase.

FIG. 15 is a graph of pH versus time for a reaction of carbon dioxideconversion to carbonic acid catalyzed with carbonic anhydraseimmobilized in tetramethyl diamine aminated polysulfone particles and noenzyme as described in example 7 wherein the experiment was performed in0.5M Na₂CO₃, 50 sccm CO₂, at 4° C., and using 50 mg carbonic anhydrase.

FIG. 16 is a graph of the total CO₂ captured in terms of mmol/min/L forcarbonic anhydrase enzymes (CA) in solutions of monoethanolamine (MEA),N,N-diethylmethylamine (DMA), and N-methyldiethanolamine (MDEA).

FIG. 17 is a graph of the specific activities of carbonic anhydrase interms of μmol/min/mg for solutions of MEA, DMA, and MDEA.

FIG. 18 is a graph of pH versus time for a reaction of carbonic acid tocarbon dioxide catalyzed with carbonic anhydrase immobilized inPEGylated polysulfone, free carbonic anhydrase, and no enzyme whereinthe experiment was performed in 0.5M MEA, 200 sccm N₂, at 20° C., andusing 25 mg (0.5 mg/mL) carbonic anhydrase.

FIG. 19 is a graph of pH versus time for a reaction of carbonic acid tocarbon dioxide catalyzed with carbonic anhydrase immobilized inPEGylated polysulfone, free carbonic anhydrase, and no enzyme whereinthe experiment was performed in 0.5M MEA, 200 sccm N₂, at 50° C., andusing 25 mg (0.5 mg/mL) carbonic anhydrase.

FIG. 20 is a graph of pH versus time for a reaction of carbonic acid tocarbon dioxide catalyzed with carbonic anhydrase immobilized inPEGylated polysulfone, free carbonic anhydrase, and no enzyme whereinthe experiment was performed in 0.5M MDEA, 200 sccm N₂, at 20° C., andusing 25 mg (0.5 mg/mL) carbonic anhydrase.

FIG. 21 is a graph of pH versus time for a reaction of carbonic acid tocarbon dioxide catalyzed with carbonic anhydrase immobilized inPEGylated polysulfone, free carbonic anhydrase, and no enzyme whereinthe experiment was performed in 0.5M MDEA, 50 sccm N₂, at 50° C., andusing 25 mg (0.5 mg/mL) carbonic anhydrase.

FIG. 22 is a graph of the enzyme activity of free carbonic anhydrase(CA) in solution (0.1 mg/mL) assayed at room temperature after exposureto 70° C. for the specified time.

FIG. 23 is a graph of the enzyme activity at 70° C. for free andimmobilized unpurified bovine carbonic anhydrase II (BCA II) for thespecified time.

FIG. 24 is a graph of the enzyme activity versus time for unpurified BCAII immobilized in polysulfone grafted with polyethylene glycol(PSf-g-PEG, 22 wt % PEG; 550 Da PEG) on lava rocks tested via a pH stat.

FIG. 25 is a graph of the enzyme activity versus time for unpurified BCAII immobilized in PSf-g-PEG (38 wt % PEG; 550 Da PEG) on lava rockstested via a pH stat.

FIG. 26 is a graph of the enzyme activity at 70° C. of free andimmobilized human carbonic anhydrase IV (HCA IV) for the specified time.

FIG. 27 is a graph of the enzyme activity versus time for HCA IVimmobilized in PSf-g-PEG (40 wt % PEG; 550 Da PEG) on lava rocks.

DESCRIPTION OF THE INVENTION

The system of the invention accelerates the rate of absorption andreaction of CO₂ into the aqueous phase of a carbonate (CO₃ ²⁻) solutionto form bicarbonate (HCO₃ ⁻). The overall chemistry is as follows:

CO₃ ²⁻+H₂O+CO₂→2HCO₃ ⁻

Without enzyme, the reaction occurs in a two-step sequence:

CO₃ ²⁻+H₂O→OH⁻+HCO₃ ⁻  (1)

CO₂+OH⁻→HCO₃ ⁻  (2)

At a pH of greater than 10.5, the reaction rate may be diffusion limitedby the low solubility of CO₂ in water. At a pH of less than 10.5, thereaction rate is very slow due to the low concentration of OH⁻.

Another way to effect the hydration of CO₂ is to use carbonic anhydrase(CA) to catalyze the reaction; the enzyme catalyzed reaction has adifferent two-step sequence:

CO₂+H₂O→H⁺+HCO₃ ⁻  (3)

CO₃ ²⁻+H⁺→HCO₃ ⁻  (4)

By using CA to catalyze CO₂ hydration [reaction (3)], the rate ofconversion of CO₂ into the bicarbonate form is accelerated, particularlyat pH less than 10.5. The K_(eq) for the hydration reaction at 25° C. is1.7×10⁻³; the reaction at equilibrium favors the CO₂/H₂O side of theequation. In the presence of CA, the reaction rate of the approach toequilibrium increases by six to eight orders of magnitude. In reaction(4), the carbonate captures the proton produced in reaction (3) andcreates a driving force to produce more bicarbonate and protons. Datashowing the increased reaction rate for CO₂ hydration as evidenced bythe faster decrease in the pH of the reaction mixture is shown in FIG.1.

Similarly in a second reactor, CA catalyzes the dehydration of thebicarbonate back into CO₃ ²⁻, CO₂, and water. The carbonate can berecycled back to the first reactor where the dehydration of CO₂ occurs.For example, the chemistry for dehydration of CO₂ is as follows:

2NaHCO₃→Na₂CO₃+H₂O+CO₂  (5)

Upon heating, bicarbonate releases the CO₂ and water and forms carbonateions that can be recycled to the hydration reaction. The CA in thedehydration reactor is similar to that in the CO₂ hydration unit andshould increase the rate of this reaction. When the sodium is replacedby another cation (e.g., alkali metal, alkaline earth metal, etc.), themetal is selected so that the resulting carbonate is preferably solublein the aqueous solution. At standard temperature and pressure, CO₂ has asolubility of about 1.8 grams/liter; thus a system allowing for rapidtransfer of CO₂ to the aqueous phase is desired.

System Design

The system used to hydrate carbon dioxide gas in a gas stream to formbicarbonate ions can use a variety of reactors, including a packed bed,a fluidized bed, or a continuous stirred tank. When a packed orfluidized bed reactor is used, the gas and liquid streams entering thereactor can be in a co-current or counter current configuration. Forexample, in a co-current system, the gas and liquid streams could enterthe reactor in the form of microbubbles of gas in the liquid stream.Further, the packing of the reactors could be packing materialcomprising immobilized carbonic anhydrase; for example, the immobilizedcarbonic anhydrase could be coated on the packing material. In some ofthese embodiments the packing material has a high surface area. Further,the configuration in the reactor could be similar to a tray styledistillation column wherein the packing material includes a membranecomprising the immobilized carbonic anhydrase is oriented to maximizethe surface contact with the gas and liquid streams (e.g., by foldingthe membrane back on itself in a serpentine configuration).

In one particular system, a two unit continuous flow system can be usedto hydrate CO₂ gas to form bicarbonate ions in a CO₂ absorber anddehydrate the bicarbonate ions to CO₂, water, and carbonate ions in aCO₂ desorber. In some instances, the units have a packed tower design. Aschematic diagram of this two unit system including an absorber 10 and adesorber 12 is depicted in FIG. 2. A CO₂ gas stream 14 enters the bottomof the absorber 10, and a liquid stream 16 enters the top portion of theabsorber 10. The liquid stream 16 is distributed over the top of thepacking (not shown) in the middle portion of the absorber 10 by adistributor (not shown). The liquid stream 16 wets the surfaces of thepacking and flows downward through the absorber 10 while the CO₂ gasstream 14 flows upward through the interstices in the packingcountercurrent to the flow of the liquid. The packing provides an areaof contact between the liquid and gas phases, and includes carbonicanhydrase immobilized on its outer surface. The CO₂ in the gas stream isabsorbed by the liquid, and the treated gas stream 18 leaves the top ofthe absorber. The liquid is enriched in CO₂ as it flows down the column,bicarbonate is formed, and the treated liquid stream 20 leaves thebottom of the absorber. The treated liquid stream 20 is pumped to a topportion of the desorber 12, and is distributed by a distributor (notshown) over packing having carbonic anhydrase immobilized thereon. Thebicarbonate within the liquid stream 20 is converted to carbon dioxide,water and carbonate. Reaction rates of this reaction to produce CO₂ canbe increased by adding heat and by increasing the rate of removal of CO₂from the desorber 12 by operating at below atmospheric pressure. Thewater and carbonate can be recycled and combined with the liquid stream16 entering the absorber 10, and the carbon dioxide leaves the top ofthe desorber as gas stream 22 and can be further processed as desired.

Alternatively, the absorber can have carbonic anhydrase immobilized onstandard reactor packing materials (such as Berl saddle, Intalox saddle,Raschig ring or Pall ring packings commonly used in packed towers) andcan be contacted with a microbubble CO₂ gas and an aqueous carbonatesolution to allow for increased surface area between the gas and liquidfor transport of the CO₂ gas into the aqueous carbonate solution.

In other embodiments, the system includes a reactor 24 as shown in FIG.2B having a membrane 26 wherein a gas stream 28 containing CO₂ is incontact with a first surface 30 of the membrane and an aqueous carbonatestream 34 is on a second surface 32 of the membrane. The membrane ispermeable to at least the CO₂ gas, but is either impermeable to theaqueous carbonate stream 34 or the first surface 30 is impermeable tothe stream 34. The membrane 26 can support an immobilized carbonicanhydrase as described herein. The CO₂ gas in the gas stream 28 caninteract with the immobilized carbonic anhydrase and the stream 34 andbe converted to bicarbonate. The bicarbonate can be absorbed by thestream 34 in contact with the immobilized enzyme. The membrane materialcan be a polysaccharide, an ion exchange resin, a treated silicon oxide,a porous metal structure, a carbon rod or tube, a graphite fiber, asilica bead, a cellulose membrane, a gel matrix (e.g., a polyacrylamidegel, a poly(acryloyl morpholine) gel, a nylon mesh and the like). Highsurface area/volume membrane systems that can be used in thisconfiguration are disclosed in U.S. Pat. No. 6,524,843.

The desorber can have carbonic anhydrase immobilized on standard reactorpacking materials and a feed of bicarbonate solution from the absorber.Reaction rates of this reaction to produce CO₂ can be increased byadding heat and the removal of CO₂ from the desorber could be increaseby operating at below atmospheric pressure.

These system designs can be combined in different configurationsdepending on the specific application or gas stream to be treated. Forexample, the system specifications can be tailored to the CO₂ content ofthe feed stream and the overall purity, recovery, and contaminant levelsrequired for the product streams along with the temperature and pressurerequirements of both streams. The use of immobilized enzymes increasesthe range of system operating conditions as compared to thecorresponding free enzyme. A packed tower as described herein can beused as the absorber in conjunction with a membrane reactor as describedherein as the desorber. Alternatively, a membrane reactor as describedherein can be used as the absorber and a packed tower as describedherein can be used as the desorber.

Also, the system design can be generally as depicted in FIG. 3B. Forexample, the carbon capture process unit comprises a standard absorptionunit and a stripping (reactive distillation) unit. The core componentsof the carbon capture system (CCS) are an absorbing unit operation, astripping unit operation, and a heat exchange component between the twounit operations. Peripheral equipment could include standard controlhardware and software, flow monitoring and regulation (e.g., controlvalves, flow meters), pumps, pH monitoring (e.g., pH meters),temperature monitoring (e.g., temperature monitors), or any combinationthereof. The additional equipment could provide means for monitoring andcontrolling the process.

Carbonic Anhydrase

The carbonic anhydrase (CA) used in the systems described hereincatalyze the conversion of carbon dioxide to bicarbonate ions andprotons and the conversion of bicarbonate ions and protons to carbondioxide. Several forms of carbonic anhydrase exist in nature. Carbonicanhydrase is found in mammals, plants, algae, and bacteria. The enzymesare usually divided into three classes (e.g., alpha, beta, and gammacarbonic anhydrase). Mammalian carbonic anhydrases belong to the alphaclass, plant carbonic anhydrases belong to the beta class, and carbonicanhydrases from methane-producing bacteria that grow in hot springsbelong to the gamma class. Members of different classes do not havesequence or structural similarity, but perform the same function andrequire a zinc ion at the active site.

For mammalian carbonic anhydrase, there are at least 14 isoforms known.These mammalian CA enzymes are divided into four broad subgroupsdepending on the tissue or cellular compartment location (e.g.,cytosolic, mitochondrial, secreted, and membrane-associated). The CAknown to have the fastest turnover rate is CA II. CA IV is known to haveparticularly high temperature stability and this stability is believedto stem from the two disulfide linkages in the enzyme.

In some of the preferred embodiments, bovine carbonic anhydrase II orhuman carbonic anhydrase IV is used. Human carbonic anhydrase IV isavailable from William S. Sly at St. Louis University and is describedin more detail in the following references: T. Okuyama, S Sato, X. L.Zhu, A. Waheed, and W. S. Sly, Human carbonic anhydrase IV: cDNAcloning, sequence comparison, and expression in COS cell membranes,Proc. Natl. Acad. Sci. USA 1992, 89(4), 1315-1319 and T. Stams, S. K.Nair, T. Okuyama, A. Waheed, W. S. Sly, D. W. Christianson, Crystalstructure of the secretory form of membrane-associated human carbonicanhydrase IV at 2.8-Å resolution, Proc. Natl. Acad. Sci. USA 1996, 93,13589-13594.

Compounds that mimic the active site of carbonic anhydrase can also beused. For example, various metal complexes have been used to mimic thecarbonic anhydrase active site. For example,[Zn₂(3,6,9,12,20,23,26,29-octaazatricyclo[29.3.1.1^(14,18)]hexatriaconta-[(34),14,16,18(36),31(35),32-hexaene)(CO₃)]Br₂.7H₂O and[Zn₂(3,6,9,12,20,23,26,29-octaazatricyclo[29.3.1.1^(14,18)]hexatriaconta-1(34),14,16,18(36),31(35),32-hexaene)(CO₃)]Br₂.0.5CH₃COCH₃.5H₂O (See Qi etal., Inorganic Chemistry Communications 2008, 11, 929-934). Also used asa mimic for carbonic anhydrase was[tris(2-benzimidazolylmethyl)amineZn(OH)₂]²⁺,[tris(2-benzimidazolyl)amineZn(OH)₂](ClO₄)₂, and[tris(hydroxy-2-benzimidazolylmethyl)amineZn(OH)₂]ClO₄.1.5H₂O were alsoused to hydrate CO₂. (See Nakata et al., The Chemistry Letters, 1997,991-992 and Echizen et al., Journal of Inorganic Biochemistry 2004, 98,1347-1360)

Enzymes and Enzyme Modifications

Enzymes including carbonic anhydrase or other enzymes can be modifiedand immobilized using the methods and material described herein. Anenzyme is used to catalyze a desired reaction. Generally,naturally-occurring enzymes, man-made enzymes, artificial enzymes andchemically or genetically modified naturally-occurring enzymes can beimmobilized. In addition, engineered enzymes that have been engineeredby natural or directed evolution can be used. Stated another way, anorganic or inorganic molecule that mimics an enzyme's properties can beused in embodiments of the present invention. The enzymes that can beimmobilized are oxidoreductases, transferases, hydrolases, lyases,isomerases, ligases, or combinations thereof. Other enzymes that can beused can be obtained by commonly used recombinant genetic methods suchas error-prone PCR and gene shuffling. Furthermore, other suitableenzymes may be obtained by the mining of enzymes from variousenvironments such as in soil. Additionally, new enzymes and forms ofenzymes can be found in microorganisms or other living sources in theenvironment.

In various preferred embodiments, enzymes immobilized are lipases,glucose isomerases, nitrilases, glucose oxidases, proteases (e.g.,pepsin), amylases (e.g., fungal amylase, maltogenic amylase),cellulases, lactases, esterases, carbohydrases, hemicellulases,pentosanases, xylanases, pullulanases, β-glucanases, acetolactatedecarboxylases, β-glucosidases, glutaminases, penicillin acylases,chloroperoxidases, aspartic β-decarboxylases, cyclodextringlycosyltransferases, subtilisins, aminoacylases, alcoholdehydrogenases, amino acid oxidases, phospholipases, ureases,cholesterases, desulfinases, lignin peroxidases, pectinases,oxidoreductases, dextranases, glucosidases, galactosidases,glucoamylases, maltases, sucrases, invertases, naringanases, bromelain,ficin, papain, pepsins, peptidases, chymosin, thermolysins, trypsins,triglyceridases, pregastric esterases, phosphatases, phytases, amidases,glutaminases, lysozyme, catalases, dehydrogenases, peroxidases, lyases,fumarases, histidases, aminotransferases, ligases, cyclases, racemases,mutases, oxidases, reductases, ligninases, laccases, listed above,haloperoxidases, hydrogenases, nitrogenases, oxynitrilases(mandelonitrile lyases), or combinations thereof.

In various embodiments, the enzyme catalyzes reactions wherein glucoseis produced. In one system, β-glucosidase can be used to hydrolyzecellobiose to glucose. Further, cellulases catalyze the hydrolysis ofcellulose to glucose and amylases catalyze the hydrolysis of starch ormaltose to glucose. Complex carbohydrates are the most abundantbiological molecules and are a good source of substrate, but glucose hasa wider range of uses than complex carbohydrates, so the carbohydratesare preferably broken down to low-molecular weight components, likeglucose. Cellulose is the most abundant complex carbohydrate and it isformed from glucose sub-units. It is easily broken down by cellulasesthat hydrolyze the glycosidic bonds. Bioreforming of complex substratesto their low-molecular weight components can be achieved by catalysiswith enzymes. These enzymes can be used for the digestion ofpolysaccharides (starch and cellulose) and disaccharides (sucrose andlactose) to individual carbohydrates that can be used in a larger numberof reactions.

In other preferred embodiments, a carbonic anhydrase can be immobilized.Carbonic anhydrase can be used to catalyze the conversion of carbondioxide to carbonic acid (e.g., bicarbonate and a proton in aqueoussolution) or the conversion of bicarbonate and a proton to carbondioxide.

For purposes of this application, the term “modification” means thatvarious functional groups on the enzyme's surface interact covalently,ionically, or by hydrophobic or hydrophilic association with variousmodifying agents. Covalent modifications to various enzymes can be madeby reaction of the enzyme with a hydrophobic agent, a hydrophilic agent,or an amphiphilic agent. These interactions add a hydrophobic,hydrophilic, or amphiphilic moiety to the enzyme. Various hydrophobicagents can be used, for example, a monoamine (e.g., alkyl amine), analdehyde (e.g., pentanal, isobutanal, acetanal, hexanal, octanal,decanal), a quaternary ammonium salt, an alkyltrimethylammonium cation,an organic cation, a phosphonium cation, a pyridinium cation, animidazolium cation, a viologen, a bis(triphenylphosphine)iminium metalcomplex, a bipyridyl metal complex, a phenanthroline-based metalcomplex, or a combination thereof. In various embodiments, thehydrophobic agent can be butyl amine, hexyl amine, octyl amine, decylamine, dodecyl amine, pentanal, isobutanal, acetanal, hexanal, octanal,decanal, acetyltrimethylammonium bromide, sodium dodecyl sulfate,ammonium lauryl sulfate, triphenylphosphonium, hexadecylpyridinium,ethidium, methyl viologen, benzyl viologen, [Ru(bipyridine)₃]²⁺,[Fe(phenanthroline)₃]³⁺, or a combination thereof. In other embodiments,the hydrophobic agent can be butyl amine, hexyl amine, octyl amine,decyl amine, dodecyl amine, pentanal, isobutanal, acetanal, hexanal,octanal, decanal, acetyltrimethylammonium bromide, sodium dodecylsulfate, ammonium lauryl sulfate, triphenylphosphonium,hexadecylpyridinium, ethidium, methyl viologen, benzyl viologen, or acombination thereof.

Further, hydrophilic agents can be used, for example, a diamine (e.g.,ethylene diamine), a monocarboxylate, a diacid (e.g., suberic acid), apolyal, a polysaccharide, a polyacrylate, a polyacrylamide, a glycosyl,an anhydride (e.g., succinic anhydride, pyromellitic anhydride, glycericaldehyde), a polyethylene glycol, agarose, or a combination thereof.Also, various amphiphilic modifying agents can be used, for example, anamino acid, fatty acids, fatty alcohols, lipids, alkyl polyethyleneoxide, other polyethylene oxide copolymers, alkyl polyglucosides, or acombination thereof. Further, the surface active agents described belowcould be used to modify the enzymes as well. The agents for covalentmodification have a functional group that is or can be made reactivewith a functional group of the enzyme being modified. Further, theenzyme can be glycosylated by using an appropriate expression system orby in vitro glycosylation wherein a saccharide moiety is attached to theenzyme.

In various preferred embodiments, the enzyme is carbonic anhydrasewherein the enzyme has been covalently modified with either an alkylamine or a water soluble polymer such as polyethylene glycol, anethylene glycol/propylene glycol copolymer, carboxymethylcellulose,dextran, polyvinyl alcohol, and the like. Alkyl amines useful forcovalent modification are butyl amine, hexyl amine, octyl amine, decylamine, dodecyl amine, and the like.

The enzymes may be modified at random positions within the molecule, orat predetermined positions within the molecule and may include one, two,three or more attached chemical moieties.

The water soluble polymer can be of any molecular weight, and can bebranched or unbranched. For polyethylene glycol, the preferred molecularweight is between about 1 kDa and about 100 kDa (the term “about”indicating that in preparations of polyethylene glycol, some moleculeswill weigh more, some less, than the stated molecular weight) for easein handling and manufacturing. Other sizes may be used, depending on thedesired properties, particularly, biological activity. For example, thepolyethylene glycol may have a mass average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 50,000, 55,000,60,000, 65,000, 70,000,75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa. In variousembodiments, the polyethylene glycol can have a mass average molecularweight from about 200 Da to about 900 Da, from about 300 Da to about 800Da, from about 400 Da to about 700 Da, from about 500 Da to about 600Da, or a mass average molecular weight of about 550 Da.

As noted above, the polyethylene glycol may have a branched structure.Branched polyethylene glycols are described, for example, in U.S. Pat.No. 5,643,575; Morpurgo et al, Appl. Biochem. Biotechnol. 56:59-72(1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999);and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosuresof each of which are incorporated herein by reference.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the enzyme with consideration of effects on functionaldomains of the enzyme. There are a number of attachment methodsavailable to those skilled in the art, e.g., EP 0 401 384, hereinincorporated by reference (coupling PEG to G-CSF), see also Malik etal., Exp. Hematol. 20:1028-1035 (1992) (reporting PEGylation of GM-CSFusing tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine, arginine,asparagine, and glutamine residues and the N-terminal amino acidresidues; those having a free carboxyl group may include aspartic acidresidues, glutamic acid residues and the C-terminal amino acid residue.Sulfhydryl groups may also be used as a reactive group for attaching thepolyethylene glycol molecules.

As suggested above, polyethylene glycol may be attached to enzymes vialinkage to any of a number of amino acid residues. For example,polyethylene glycol can be linked to an enzyme via covalent bonds tolysine, histidine, aspartic acid, glutamic acid, or cysteine residues.One or more reaction chemistries may be employed to attach polyethyleneglycol to specific amino acid residues (e.g., lysine, histidine,aspartic acid, glutamic acid, or cysteine) of the enzyme or to more thanone type of amino acid residue (e.g., lysine, histidine, aspartic acid,glutamic acid, cysteine and combinations thereof) of the enzyme.

As indicated above, PEGylation of the enzymes may be accomplished by anynumber of means. For example, polyethylene glycol may be attached to theenzyme either directly or by an intervening linker. Linkerless systemsfor attaching polyethylene glycol to enzymes are described in Delgado etal., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis etal., Inter J. of Hematol. 68:1-18 (1998); U.S. Pat. No. 4,002,531; U.S.Pat. No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures ofeach of which are incorporated herein by reference.

One system for attaching polyethylene glycol directly to amino acidresidues of enzymes without an intervening linker employs tresylatedMPEG, which is produced by the modification of monomethoxy polyethyleneglycol (MPEG) using tresylchloride (ClSO₂CH₂CF₃). Upon reaction of theenzyme with tresylated MPEG, polyethylene glycol is directly attached toamine groups of the enzyme. Thus, the invention includesenzyme-polyethylene glycol conjugates produced by reacting enzymes ofthe invention with a polyethylene glycol molecule having a2,2,2-trifluoroethane sulfonyl group.

Polyethylene glycol can also be attached to enzymes using a number ofdifferent intervening linkers. For example, U.S. Pat. No. 5,612,460, theentire disclosure of which is incorporated herein by reference,discloses urethane linkers for connecting polyethylene glycol toenzymes. Enzyme-polyethylene glycol conjugates wherein the polyethyleneglycol is attached to the enzyme by a linker can also be produced byreaction of enzymes with compounds such as MPEG-succinimidylsuccinate,MPEG activated with 1,1′-carbonyldiimidazole,MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, andvarious MPEG-succinate derivatives. A number of additional polyethyleneglycol derivatives and reaction chemistries for attaching polyethyleneglycol to enzymes are described in WO 98/32466, the entire disclosure ofwhich is incorporated herein by reference.

The number of polyethylene glycol moieties attached to each enzyme(i.e., the degree of substitution) may also vary. For example, thePEGylated enzymes may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly,the average degree of substitution ranges from 1-3, 2-4, 3-5, 4-6, 5-7,6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18,17-19, or 18-20 polyethylene glycol moieties per enzyme molecule.Methods for determining the degree of substitution are discussed, forexample, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys.9:249-304 (1992).

When an amine is used to modify the enzyme, the enzyme is combined witha coupling agent (e.g., N-(3-dimethylaminopropyl)-N-ethylcarbodiimidehydrochloride (EDC), dicyclohexylcarbodiiminde (DCC),N,N′-diisopropylcarbodiimide (DIC)) and an ester activating agent (e.g.,N-hydroxysulfosuccinimide sodium salt (Sulfo-NHS)),1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotrizole); the resultingsolution is vigorously vortexed for five seconds. A second solution ismade with a MES buffer of pH 5.0 combined with an alkyl amine or apolyethylene glycol. This solution is combined with the couplingagent/enzyme solution and vigorously vortexed for 5 seconds. Thecombined solutions are held refrigerated overnight. Then the modifiedenzyme can be immobilized in the immobilization materials describedherein.

Further, the enzymes can be modified by various surface active agents.For example, non-ionic surface active agents can beN,N-bis(3-D-gluconamidopropyl)cholamide (BigCHAP),N,N-bis(3-D-gluconamidopropyl)deoxycholamide (DeoxyBigCHAP), apolyoxyethylene alcohol (e.g., Brij35 and Brij 58 P),2-cyclohexylmethyl-β-D-maltoside (Cymal-1),2-cyclohexylethyl-β-D-maltoside (Cymal-2),cyclohexylpentyl-3-D-maltoside (Cymal-5),cyclohexylhexyl-β-D-maltoside(Cymal-6), decyl-β-D-maltopyranoside,n-dodecyl-β-D-maltoside, n-hexyadecyl-β-D-maltoside,undecyl-β-D-maltoside, decyl-β-D-1-thiomaltopyranoside,octyl-3-D-thioglucopyranoside, digitonin, dimethydecylphosphine oxide,dodecyldimethylphosphine oxide, (octylphenoxy)polyethoxyethanol (IGEPAL®CA630), N-octanoyl-N-methylglucamine (MEGA-8),N-nonanoyl-N-methylglucamine (MEGA-9), N-decanoyl-N-methylglucamine(MEGA-10), a polyoxy ethylene octyl phenol (Nonidet® P40-substitute), apolyoxyethylene-polyoxypropylene block co-polymer (Pluronic F-68),saponin, polyoxyethylene 9-lauryl ether (Thesit®), a polyoxy ethyleneoctyl phenol (e.g., Triton® X-100 and Triton® X-114), a polyoxyethylenederivative of sorbitan monolaurate (e.g., TWEEN® 20, TWEEN® 40, andTWEEN® 80), N,N-dimethyldodecylamine-N-oxide, an alcohol ethoxylate(Synperonic A7), or a combination thereof.

Zwitterionic surface active agents can also be used, for exampleamidosulfobetaine-14, amidosulfobetaine-16, C7BzO,3-[(3-cholamidopropyldimethylammonio]-1-propanesulphonate (CHAPS),3-[(3-cholamidopropyldimethylammonio]-2-hydroxy-1-propanesulphonate(CHAPSO), (dodecyldimethylammonio)acetate (EMPIGEN® BB),3-(N,N-dimethyloctylammonio) propanesulfonate,3-(dodecylammonio)propanesulfonate, 3-(N,N-dimethylmyristylammonio)propanesulfonate, 3-(N,N-dimethylpalmitylammonio) propanesulfonate,3-(N,N-dimethyloctadecylammonio) propanesulfonate, or a combinationthereof.

When the enzyme is modified with a surface active agent, the modifiedenzyme is prepared by combining the enzyme with a surface active agentin a buffer of the appropriate pH for the enzyme. One of ordinary skillin the art could readily determine buffers of appropriate pH for aparticular enzyme.

Enzyme Immobilization Materials

For purposes of the present invention, an enzyme is “stabilized” if iteither: (1) retains at least about 15% of its initial catalytic activityfor at least about 30 days when continuously catalyzing a chemicaltransformation at room temperature; (2) retains at least about 15% ofits initial catalytic activity for at least about 5 days whencontinuously catalyzing a chemical transformation at room temperature;(3) retains at least about 15% of its initial catalytic activity for atleast about 5 days when being treated at temperatures from about 30° C.to about 100° C., (4) retains at least about 15% of its initialcatalytic activity for at least about 5 days when continuouslycatalyzing a chemical transformation at room temperature and a pH fromabout 0 to about 13, (5) retains at least about 15% of its initialcatalytic activity for at least about 5 days when continuouslycatalyzing a chemical transformation at room temperature in a non-polarsolvent, an oil, an alcohol, acetonitrile, or a high ion concentration.Typically, a free enzyme in solution loses its catalytic activity withina few hours to a few days, whereas a properly immobilized and stabilizedenzyme can retain its catalytic activity for at least about 5 days toabout 1095 days (3 years). Thus, the immobilization of the enzymeprovides a significant advantage in stability. The retention ofcatalytic activity is defined as the enzyme having at least about 15% ofits initial activity, which can be measured by a means that demonstrateenzyme-mediated generation of product such as chemiluminescence,electrochemical, mass spectrometry, spectrophotometric (i.e. UV-Vis),radiochemical, or fluorescence assay wherein the intensity of theproperty is measured at an initial time. In various embodiments, theenzyme retains at least about 15% of its initial activity while theenzyme is continuously catalyzing a chemical transformation.

With respect to the stabilization of the enzyme, the enzymeimmobilization material provides a chemical and/or mechanical barrier toprevent or impede enzyme denaturation. To this end, the enzymeimmobilization material physically confines the enzyme, preventing theenzyme from unfolding. The process of unfolding an enzyme from a foldedthree-dimensional structure is one mechanism of enzyme denaturation.

In some embodiments, the enzyme immobilization material stabilizes theenzyme so that the enzyme retains its catalytic activity for at leastabout 5 days to about 730 days (2 years). In other embodiments, theimmobilized enzyme retains at least about 75% of its initial catalyticactivity for at least about 30, 45, 60, 75, 90, 105, 120, 150, 180, 210,240, 270, 300, 330, 365, 400, 450, 500, 550, 600, 650, 700, 730, 800,850, 900, 950, 1000, 1050, 1095 days or more. In some instances, theimmobilized enzyme retains about 75% to about 95% of its initialcatalytic activity for about 30 to about 1095 days, about 45 to about1095 days, about 60 to about 1095 days, about 75 to about 1095 days,about 90 to about 1095 days, about 105 to about 1095 days, about 120 toabout 1095 days, about 150 to about 1095 days, about 180 to about 1095days, about 210 to about 1095 days, about 240 to about 1095 days, about270 to about 1095 days, about 300 to about 1095 days, about 330 to about1095 days, about 365 to about 1095 days, about 400 to about 1095 days,about 450 to about 1095 days, about 500 to about 1095 days, about 550 toabout 1095 days, about 600 to about 1095 days, about 650 to about 1095days, about 700 to about 1095 days, or about 730 to about 1095 days. Invarious embodiments, the immobilized enzyme retains at least about 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% ormore of its initial catalytic activity for at least about 5, 7, 10, 15,20, 25, 30, 45, 60, 75, 90, 105, 120, 150, 180, 210, 240, 270, 300, 330,365, 400, 450, 500, 550, 600, 650, 700, 730, 800, 850, 900, 950, 1000,1050, 1095 days or more. In some instances, the immobilized enzymeretains about 15 to about 95%, about 20 to about 95%, about 25 to about95%, about 30 to about 95%, about 35 to about 95%, about 40 to about95%, about 45 to about 95%, about 50 to about 95%, about 55 to about95%, about 60 to about 95%, about 65 to about 95%, about 70 to about95%, about 75 to about 95%, about 80 to about 95%, about 85 to about95%, or about 90 to about 95% of its initial catalytic activity forabout 5 to about 1095 days, about 7 to about 1095 days, about 10 toabout 1095 days, about 15 to about 1095 days, about 20 to about 1095days, about 25 to about 1095 days, about 30 to about 1095 days, about 45to about 1095 days, about 60 to about 1095 days, about 75 to about 1095days, about 90 to about 1095 days, about 105 to about 1095 days, about120 to about 1095 days, about 150 to about 1095 days, about 180 to about1095 days, about 210 to about 1095 days, about 240 to about 1095 days,about 270 to about 1095 days, about 300 to about 1095 days, about 330 toabout 1095 days, about 365 to about 1095 days, about 400 to about 1095days, about 450 to about 1095 days, about 500 to about 1095 days, about550 to about 1095 days, about 600 to about 1095 days, about 650 to about1095 days, about 700 to about 1095 days, or about 730 to about 1095days.

In various embodiments, an enzyme having greater temperature or pHstability may also retain at least about 75% of its initial catalyticactivity for at least about 5 days when actively catalyzing a chemicaltransformation as described above.

In other embodiments, when exposed to a pH of less than about 2, lessthan about 3, less than about 4, or less than about 5, the stabilizedenzyme retains at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90 or 95% of its initial catalytic activity for atleast about 5, 10, 15, 30, 40, 50, 60, 75, 90 days or more whencontinuously catalyzing a chemical transformation. In some instances,when exposed to a pH of less than about 2, less than about 3, less thanabout 4, or less than about 5, the stabilized enzyme retains about 15 toabout 95%, about 20 to about 95%, about 25 to about 95%, about 30 toabout 95%, about 35 to about 95%, about 40 to about 95%, about 45 toabout 95%, about 50 to about 95%, about 55 to about 95%, about 60 toabout 95%, about 65 to about 95%, about 70 to about 95%, about 75 toabout 95%, about 80 to about 95%, about 85 to about 95%, or about 90 toabout 95% of its initial catalytic activity for about 5 to 90 days,about 10 to 90 days, about 15 to 90 days, about 20 to 90 days, about 25to 90 days, about 30 to 90 days, about 35 to 90 days, about 40 to 90days, about 45 to 90 days, about 50 to 90 days, about 55 to 90 days,about 60 to 90 days, about 65 to 90 days, about 70 to 90 days, about 75to 90 days, about 80 to 90 days, about 85 to 90 days when continuouslycatalyzing a chemical transformation. In some instances, when exposed toa pH of less than about 2, less than about 3, less than about 4, or lessthan about 5, the stabilized enzyme retains about 15 to about 95%, about20 to about 95%, about 25 to about 95%, about 30 to about 95%, about 35to about 95%, about 40 to about 95%, about 45 to about 95%, about 50 toabout 95%, about 55 to about 95%, about 60 to about 95%, about 65 toabout 95%, about 70 to about 95%, about 75 to about 95%, about 80 toabout 95%, about 85 to about 95%, or about 90 to about 95% of itsinitial catalytic activity for at least about 5, 10, 15, 30, 40, 50, 60,75, 90 days or more when continuously catalyzing a chemicaltransformation. In some instances, when exposed to a pH of greater thanabout 9, greater than about 10, greater than about 11, or greater thanabout 12, the stabilized enzyme retains about 15 to about 95%, about 20to about 95%, about 25 to about 95%, about 30 to about 95%, about 35 toabout 95%, about 40 to about 95%, about 45 to about 95%, about 50 toabout 95%, about 55 to about 95%, about 60 to about 95%, about 65 toabout 95%, about 70 to about 95%, about 75 to about 95%, about 80 toabout 95%, about 85 to about 95%, or about 90 to about 95% of itsinitial catalytic activity for about 5 to 90 days, about 10 to 90 days,about 15 to 90 days, about 20 to 90 days, about 25 to 90 days, about 30to 90 days, about 35 to 90 days, about 40 to 90 days, about 45 to 90days, about 50 to 90 days, about 55 to 90 days, about 60 to 90 days,about 65 to 90 days, about 70 to 90 days, about 75 to 90 days, about 80to 90 days, about 85 to 90 days when continuously catalyzing a chemicaltransformation.

In other embodiments, when exposed to an agent such as a nonpolarsolvent, an oil, an alcohol, acetonitrile, a concentrated ionicsolution, or combination thereof, the stabilized enzyme retains at leastabout 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or95% of its initial catalytic activity for at least about 5, 10, 15, 30,40, 50, 60, 75, 90 days or more when continuously catalyzing a chemicaltransformation. In some instances, when exposed to the agent, thestabilized enzyme retains about 10 to about 95%, about 15 to about 95%,about 20 to about 95%, about 25 to about 95%, about 30 to about 95%,about 35 to about 95%, about 40 to about 95%, about 45 to about 95%,about 50 to about 95%, about 55 to about 95%, about 60 to about 95%,about 65 to about 95%, about 70 to about 95%, about 75 to about 95%,about 80 to about 95%, about 85 to about 95%, or about 90 to about 95%of its initial catalytic activity for about 5 to 90 days, about 10 to 90days, about 15 to 90 days, about 20 to 90 days, about 25 to 90 days,about 30 to 90 days, about 35 to 90 days, about 40 to 90 days, about 45to 90 days, about 50 to 90 days, about 55 to 90 days, about 60 to 90days, about 65 to 90 days, about 70 to 90 days, about 75 to 90 days,about 80 to 90 days, about 85 to 90 days when continuously catalyzing achemical transformation. In these instances, the concentration of theagent can be from about 1 wt. % to about 95 wt. %, 5 wt. % to about 95wt. %, 10 wt. % to about 95 wt. %, 15 wt. % to about 95 wt. %, 20 wt. %to about 95 wt. %, 30 wt. % to about 95 wt. %, 40 wt. % to about 95 wt.%, 50 wt. % to about 95 wt. %.

An immobilized enzyme is an enzyme that is physically confined in acertain region of the enzyme immobilization material while retaining itscatalytic activity. There are a variety of methods for enzymeimmobilization, including carrier-binding, cross-linking and entrapping.Carrier-binding is the binding of enzymes to water-insoluble carriers.Cross-linking is the intermolecular cross-linking of enzymes bybifunctional or multifunctional reagents. Entrapping is incorporatingenzymes into the lattices of a semipermeable material. The particularmethod of enzyme immobilization is not critically important, so long asthe enzyme immobilization material (1) immobilizes the enzyme, and insome embodiments, (2) stabilizes the enzyme. In various embodiments, theenzyme immobilization material is also permeable to a compound smallerthan the enzyme. An enzyme is adsorbed to an immobilization materialwhen it adheres to the surface of the material by chemical or physicalinteractions. Further, an enzyme is immobilized by entrapment when theenzyme is contained within the immobilization material whether within apocket of the material or not.

With reference to the immobilization material's permeability to variouscompounds that are smaller than an enzyme, the immobilization materialallows the movement of a substrate compound through it so the substratecompound can contact the enzyme. The immobilization material can beprepared in a manner such that it contains internal pores, micellarpockets, channels, openings or a combination thereof, which allow themovement of the substrate compound throughout the immobilizationmaterial, but which constrain the enzyme to substantially the same spacewithin the immobilization material. Such constraint allows the enzyme toretain its catalytic activity. In various preferred embodiments, theenzyme is confined to a space that is substantially the same size andshape as the enzyme, wherein the enzyme retains substantially all of itscatalytic activity. The pores, micellar pockets, channels, or openingshave physical dimensions that satisfy the above requirements and dependon the size and shape of the specific enzyme to be immobilized.

In some of the embodiments, the enzyme is preferably located within apore of the immobilization material and the compound travels in and outof the immobilization material through transport channels. The pores ofthe enzyme immobilization material can be from about 6 nm to about 30nm, from about 10 nm to about 30 nm, from about 15 nm to about 30 nm,from about 20 nm to about 30 nm, from about 25 nm to about 30 nm, fromabout 6 nm to about 20 nm, or from about 10 nm to about 20 nm. Therelative size of the pores and transport channels can be such that apore is large enough to immobilize an enzyme, but the transport channelsare too small for the enzyme to travel through them. Further, atransport channel preferably has a diameter of at least about 10 nm. Insome embodiments, the pore diameter to transport channel diameter ratiois at least about 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1,6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1 or more; the porediameter to transport channel diameter ratio can be about 2:1 to about10:1, about 2.5:1 to about 10:1, about 3:1 to about 10:1, about 3.5:1 toabout 10:1, about 4:1 to about 10:1, about 4.5:1 to about 10:1, about5:1 to about 10:1, about 5.5:1 to about 10:1, about 6:1 to about 10:1,about 6.5:1 to about 10:1, about 7:1 to about 10:1, about 7.5:1 to about10:1, about 8:1 to about 10:1, about 8.5:1 to about 10:1, about 9:1 toabout 10:1, or about 9.5:1 to about 10:1. In yet another embodiment,preferably, a transport channel has a diameter of at least about 2 nmand the pore diameter to transport channel diameter ratio is at leastabout 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1,7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1 or more; the pore diameter totransport channel diameter ratio can be about 2:1 to about 10:1, about2.5:1 to about 10:1, about 3:1 to about 10:1, about 3.5:1 to about 10:1,about 4:1 to about 10:1, about 4.5:1 to about 10:1, about 5:1 to about10:1, about 5.5:1 to about 10:1, about 6:1 to about 10:1, about 6.5:1 toabout 10:1, about 7:1 to about 10:1, about 7.5:1 to about 10:1, about8:1 to about 10:1, about 8.5:1 to about 10:1, about 9:1 to about 10:1,or about 9.5:1 to about 10:1.

In some of the various embodiments, when the enzyme is large oraggregated, the enzyme immobilization material can have a pore size thatis substantially the same size as the enzyme or aggregated enzyme. Suchan enzyme immobilization material can have pores that constrain theenzyme or aggregated enzyme in substantially the same space within theenzyme immobilization material and allow diffusion of compounds that aresmaller than the enzyme or aggregated enzyme through the material. Thisenzyme immobilization material would have an average micelle size offrom about 15 nm to about 2000 nm, from about 50 nm to about 2000 nm,from about 100 nm to about 2000 nm, from about 200 nm to about 2000 nm,from about 300 nm to about 2000 nm, from about 400 nm to about 2000 nm,from about 500 nm to about 2000 nm, from about 600 nm to about 2000 nm,from about 700 nm to about 2000 nm, from about 800 nm to about 2000 nm,from about 20 nm to about 1000 nm, from about 50 nm to about 1000 nm,from about 100 nm to about 1000 nm, from about 200 nm to about 1000 nm,from about 300 nm to about 1000 nm, from about 400 nm to about 1000 nm,from about 500 nm to about 1000 nm, from about 600 nm to about 1000 nm,or from about 700 nm to about 1000 nm.

In some of these embodiments, the immobilization material has a micellaror inverted micellar structure. Generally, the molecules making up amicelle are amphipathic, meaning they contain a polar, hydrophilic groupand a nonpolar, hydrophobic group. The molecules can aggregate to form amicelle, where the polar groups are on the surface of the aggregate andthe hydrocarbon, nonpolar groups are sequestered inside the aggregate.Inverted micelles have the opposite orientation of polar groups andnonpolar groups. The amphipathic molecules making up the aggregate canbe arranged in a variety of ways so long as the polar groups are inproximity to each other and the nonpolar groups are in proximity to eachother. Also, the molecules can form a bilayer with the nonpolar groupspointing toward each other and the polar groups pointing away from eachother. Alternatively, a bilayer can form wherein the polar groups canpoint toward each other in the bilayer, while the nonpolar groups pointaway from each other.

Modified Nafion®

In one preferred embodiment, the micellar immobilization material is amodified perfluoro sulfonic acid-PTFE copolymer (or modifiedperfluorinated ion exchange polymer)(modified Nafion® or modifiedFlemion®) membrane. The perfluorinated ion exchange polymer membrane ismodified with a hydrophobic cation that is larger than the ammonium (NH₄⁺) ion. The hydrophobic cation serves the dual function of (1) dictatingthe membrane's pore size and (2) acting as a chemical buffer to helpmaintain the pore's pH level, both of which stabilize the enzyme.

With regard to the first function of the hydrophobic cation,mixture-casting a perfluoro sulfonic acid-PTFE copolymer (orperfluorinated ion exchange polymer) with a hydrophobic cation toproduce a modified perfluoro sulfonic acid-PTFE copolymer (or modifiedperfluorinated ion exchange polymer)(Nafion® or Flemion®) membraneprovides an immobilization material wherein the pore size is dependenton the size of the hydrophobic cation. Accordingly, the larger thehydrophobic cation, the larger the pore size. This function of thehydrophobic cation allows the pore size to be made larger or smaller tofit a specific enzyme by varying the size of the hydrophobic cation.

Regarding the second function of the hydrophobic cation, the propertiesof the perfluoro sulfonic acid-PTFE copolymer (or perfluorinated ionexchange polymer) membrane are altered by exchanging the hydrophobiccation for protons as the counterion to the —SO₃ ⁻ groups on theperfluoro sulfonic acid-PTFE copolymer (or anions on the perfluorinatedion exchange polymer) membrane. This change in counterion provides abuffering effect on the pH because the hydrophobic cation has a muchgreater affinity for the —SO₃ ⁻ sites than protons do. This bufferingeffect of the membrane causes the pH of the pore to remain substantiallyunchanged with changing solution pH; stated another way, the pH of thepore resists changes in the solution's pH. In addition, the membraneprovides a mechanical barrier, which further protects the immobilizedenzymes.

In order to prepare a modified perfluoro sulfonic acid-PTFE copolymer(or perfluorinated ion exchange polymer) membrane, the first step is tocast a suspension of perfluoro sulfonic acid-PTFE copolymer (orperfluorinated ion exchange polymer), particularly Nafion®, with asolution of the hydrophobic cations to form a membrane. The excesshydrophobic cations and their salts are then extracted from themembrane, and the membrane is re-cast. Upon re-casting, the membranecontains the hydrophobic cations in association with the —SO₃ ⁻ sites ofthe perfluoro sulfonic acid-PTFE copolymer (or perfluorinated ionexchange polymer) membrane. Removal of the salts of the hydrophobiccation from the membrane results in a more stable and reproduciblemembrane; if they are not removed, the excess salts can become trappedin the pore or cause voids in the membrane.

In one embodiment, a modified Nafion® membrane is prepared by casting asuspension of Nafion® polymer with a solution of a salt of a hydrophobiccation such as quaternary ammonium bromide. Excess quaternary ammoniumbromide or hydrogen bromide is removed from the membrane before it isre-cast to form the salt-extracted membrane. Salt extraction ofmembranes retains the presence of the quaternary ammonium cations at thesulfonic acid exchange sites, but eliminates complications from excesssalt that may be trapped in the pore or may cause voids in theequilibrated membrane. The chemical and physical properties of thesalt-extracted membranes have been characterized by voltammetry, ionexchange capacity measurements, and fluorescence microscopy beforeenzyme immobilization. Exemplary hydrophobic cations are ammonium-basedcations, quaternary ammonium cations, alkyltrimethylammonium cations,alkyltriethylammonium cations, organic cations, phosphonium cations,triphenylphosphonium, pyridinium cations, imidazolium cations,hexadecylpyridinium, ethidium, viologens, methyl viologen, benzylviologen, bis(triphenylphosphine)iminium, metal complexes, bipyridylmetal complexes, phenanthroline-based metal complexes,[Ru(bipyridine)₃]²⁺ and [Fe(phenanthroline)₃]³⁺.

In one preferred embodiment, the hydrophobic cations are ammonium-basedcations. In particular, the hydrophobic cations are quaternary ammoniumcations. In another embodiment, the quaternary ammonium cations arerepresented by Formula 1:

wherein R₁, R₂, R₃, and R₄ are independently hydrogen, hydrocarbyl,substituted hydrocarbyl, or heterocyclo wherein at least one of R₁, R₂,R₃, and R₄ is other than hydrogen. In a further embodiment, preferably,R₁, R₂, R₃, and R₄ are independently hydrogen, methyl, ethyl, propyl,butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl,tridecyl or tetradecyl wherein at least one of R₁, R₂, R₃, and R₄ isother than hydrogen. In still another embodiment, R₁, R₂, R₃, and R₄ arethe same and are methyl, ethyl, propyl, butyl, pentyl or hexyl. In yetanother embodiment, preferably, R₁, R₂, R₃, and R₄ are butyl. In yetanother embodiment, preferably, R₁, R₂, R₃, and R₄ are ethyl.Preferably, the quaternary ammonium cation is tetraethylammonium (T2A),tetrapropylammonium (T3A), tetrapentylammonium (T5A), tetrahexylammonium(T6A), tetraheptylammonium (T7A), trimethylicosylammonium (TMICA),trimethyloctyldecylammonium (TMODA), trimethylhexyldecylammonium(TMHDA), trimethyltetradecylammonium (TMTDA), trimethyloctylammonium(TMOA), trimethyldodecylammonium (TMDDA), trimethyldecylammonium (TMDA),trimethylhexylammonium (TMHA), tetrabutylammonium (TBA),triethylhexylammonium (TEHA), and combinations thereof.

Carbonic anhydrase can be immobilized in TEAB-modified Nafion® asfollows. Tetraethyl ammonium bromide (TEAB) modified Nafion® is added toethanol to make a solution having a concentration of 5.0 wt. %. Thecarbonic anhydrase is added to a buffer solution and a surface activeagent is added at a total solution percentage of 0.5% and stirred untila uniform dissolution occurs. Once the solution is adequately dispersed,the TEAB-modified Nafion® solution is added and stirred until thesolution is sufficiently homogenous. Once the immobilized enzymesolution is thoroughly mixed, it is cast onto a high surface areasupport and allowed to dry for 12 hours at 4° C. followed by two hoursunder vacuum. Alternatively, a high surface carbon support can be addedto the immobilized enzyme solution, mixed, sprayed, and allowed to dryfor several hours at room temperature.

Hydrophobically Modified Polysaccharides

In other various embodiments, exemplary micellar or inverted micellarimmobilization materials are hydrophobically modified polysaccharides,these polysaccharides are selected from chitosan, cellulose, chitin,starch, amylose, alginate, glycogen, and combinations thereof. Invarious embodiments, the micellar or inverted micellar immobilizationmaterials are polycationic polymers, particularly, hydrophobicallymodified chitosan. Chitosan is apoly[β-1-(1-4)-2-amino-2-deoxy-D-glucopyranose]. Chitosan is typicallyprepared by deacetylation of chitin (apoly[β1-(1-4)-2-acetamido-2-deoxy-D-glucopyranose]). The typicalcommercial chitosan has approximately 85% deacetylation. Thesedeacetylated or free amine groups can be further functionalized withhydrocarbyl, particularly, alkyl groups. Thus, in various embodiments,the micellar hydrophobically modified chitosan corresponds to thestructure of Formula 2

wherein n is an integer; R₁₀ is independently hydrogen, hydrocarbyl,substituted hydrocarbyl, or a hydrophobic redox mediator; and R₁₁ isindependently hydrogen, hydrocarbyl, substituted hydrocarbyl, or ahydrophobic redox mediator. In certain embodiments of the invention, nis an integer that gives the polymer a molecular weight of from about21,000 to about 4,000,000; from about 21,000 to about 500,000;preferably, from about 90,000 to about 500,000; more preferably, fromabout 150,000 to about 350,000; more preferably, from about 225,000 toabout 275,000. In many embodiments, R₁₀ is independently hydrogen oralkyl and R₁₁ is independently hydrogen or alkyl. Further, R₁₀ isindependently hydrogen or hexyl and R₁₁ is independently hydrogen orhexyl. Alternatively, R₁₀ is independently hydrogen or octyl and R₁₁ isindependently hydrogen or octyl.

In other various embodiments, the micellar hydrophobically modifiedchitosan is a micellar hydrophobic redox mediator modified chitosancorresponding to Formula 2A

wherein n is an integer; R_(10a) is independently hydrogen, or ahydrophobic redox mediator; and R_(11a) is independently hydrogen, or ahydrophobic redox mediator.

Further, in various embodiments, the micellar hydrophobically modifiedchitosan is a modified chitosan or redox mediator modified chitosancorresponding to Formula 2B

wherein R₁₁, R₁₂, and n are defined as in connection with Formula 2. Insome embodiments, R₁₁ and R₁₂ are independently hydrogen or straight orbranched alkyl; preferably, hydrogen, butyl, pentyl, hexyl, heptyl,octyl, nonyl, decyl, undecyl, or dodecyl. In various embodiments, R₁₁and R₁₂ are independently hydrogen, butyl, or hexyl.

The micellar hydrophobically modified chitosans can be modified withhydrophobic groups to varying degrees. The degree of hydrophobicmodification is determined by the percentage of free amine groups thatare modified with hydrophobic groups as compared to the number of freeamine groups in the unmodified chitosan. The degree of hydrophobicmodification can be estimated from an acid-base titration and/or nuclearmagnetic resonance (NMR), particularly ¹H NMR, data. This degree ofhydrophobic modification can vary widely and is at least about 0.25,0.5, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 32, 24, 26, 28, 40,42, 44, 46, 48%, or more. Preferably, the degree of hydrophobicmodification is from about 10% to about 45%; from about 10% to about35%; from about 20% to about 35%; or from about 30% to about 35%.

In other various embodiments, the hydrophobic redox mediator of Formula2A is a transition metal complex of osmium, ruthenium, iron, nickel,rhodium, rhenium, or cobalt with 1,10-phenanthroline (phen),2,2′-bipyridine (bpy) or 2,2′,2″-terpyridine (terpy), methylene green,methylene blue, poly(methylene green), poly(methylene blue), luminol,nitro-fluorenone derivatives, azines, osmium phenanthrolinedione,catechol-pendant terpyridine, toluene blue, cresyl blue, nile blue,neutral red, phenazine derivatives, thionin, azure A, azure B, toluidineblue O, acetophenone, metallophthalocyanines, nile blue A, modifiedtransition metal ligands, 1,10-phenanthroline-5,6-dione,1,10-phenanthroline-5,6-diol, [Re(phen-dione)(CO)₃Cl],[Re(phen-dione)₃](PF₆)₂, poly(metallophthalocyanine), poly(thionine),quinones, diimines, diaminobenzenes, diaminopyridines, phenothiazine,phenoxazine, toluidine blue, brilliant cresyl blue,3,4-dihydroxybenzaldehyde, poly(acrylic acid), poly(azure I), poly(nileblue A), polyaniline, polypyridine, polypyrole, polythiophene,poly(thieno[3,4-b]thiophene), poly(3-hexylthiophene),poly(3,4-ethylenedioxypyrrole), poly(isothianaphthene),poly(3,4-ethylenedioxythiophene), poly(difluoroacetylene),poly(4-dicyanomethylene-4H-cyclopenta[2,1-b;3,4-b′]dithiophene),poly(3-(4-fluorophenyl)thiophene), poly(neutral red), or combinationsthereof.

Preferably, the hydrophobic redox mediator is Ru(phen)₃ ⁺², Fe(phen)₃⁺², Os(phen)₃ ⁺², Co(phen)₃ ⁺², Cr(phen)₃ ⁺², Ru(bpy)₃ ⁺², Os(bpy)₃ ⁺²,Fe(bpy)₃ ⁺², Co(bpy)₃ ⁺², Cr(bpy)₃ ⁺², Os(terpy)₃ ⁺²,Ru(bpy)₂(4-methyl-4′-(6-hexyl)-2,2′-bipyridine)⁺²,Co(bpy)₂(4-methyl-4′-(6-hexyl)-2,2′-bipyridine)⁺²,Cr(bpy)₂(4-methyl-4′-(6-hexyl)-2,2′-bipyridine)⁺²,Fe(bpy)₂(4-methyl-4′-(6-hexyl)-2,2′-bipyridine)⁺²,Os(bpy)₂(4-methyl-4′-(6-hexyl)-2,2′-bipyridine)⁺², or combinationsthereof. More preferably, the hydrophobic redox mediator isRu(bpy)₂(4-methyl-4′-(6-hexyl)-2,2′-bipyridine)⁺²,Co(bpy)₂(4-methyl-4′-(6-hexyl)-2,2′-bipyridine)⁺²,Cr(bpy)₂(4-methyl-4′-(6-hexyl)-2,2′-bipyridine)⁺²,Fe(bpy)₂(4-methyl-4′-(6-hexyl)-2,2′-bipyridine)⁺²,Os(bpy)₂(4-methyl-4′-(6-hexyl)-2,2′-bipyridine)⁺², or combinationsthereof. In various preferred embodiments, the hydrophobic redoxmediator is Ru(bpy)₂(4-methyl-4′-(6-hexyl)-2,2′-bipyridine)⁺².

For the immobilization material having a hydrophobic redox mediator asthe modifier, the hydrophobic redox mediator is typically covalentlybonded to the chitosan or polysaccharide backbone. Typically, in thecase of chitosan, the hydrophobic redox mediator is covalently bonded toone of the amine functionalities of the chitosan through a —N—C— bond.In the case of metal complex redox mediators, the metal complex isattached to the chitosan through an —N—C— bond from a chitosan aminegroup to an alkyl group attached to one or more of the ligands of themetal complex. A structure corresponding to Formula 2C is an example ofa metal complex attached to a chitosan

wherein n is an integer; R_(10c) is independently hydrogen or astructure corresponding to Formula 2D; R_(11c) is independently hydrogenor a structure corresponding to Formula 1D; m is an integer from 0 to10; M is Ru, Os, Fe, Cr, or Co; and heterocycle is bipyridyl,substituted bipyridyl, phenanthroline, acetylacetone, and combinationsthereof.

The hydrophobic group used to modify chitosan serves the dual functionof (1) dictating the immobilization material's micelle size and (2)modifying the chitosan's chemical environment to maintain an acceptablemicelle environment, both of which stabilize the enzyme. With regard tothe first function of the hydrophobic group, hydrophobically modifyingchitosan produces an immobilization material wherein the pore size isdependent on the size of the hydrophobic group. Accordingly, the size,shape, and extent of the modification of the chitosan with thehydrophobic group affects the size and shape of the micellarpore/pocket. This function of the hydrophobic group allows the micellarpore/pocket size to be made larger or smaller or a different shape tofit a specific enzyme by varying the size and branching of thehydrophobic group.

Regarding the second function of the hydrophobic cation, the propertiesof the hydrophobically modified chitosan membranes are altered bymodifying chitosan with hydrophobic groups. This hydrophobicmodification of chitosan affects the pore environment by increasing thenumber of available exchange sites to proton. In addition to affectingthe pH of the material, the hydrophobic modification of chitosanprovides a membrane that is a mechanical barrier, which further protectsthe immobilized enzymes.

Table 1 shows the number of available exchange sites to proton for thehydrophobically modified chitosan membrane.

TABLE 1 Number of available exchange sites to proton per gram ofchitosan polymer Exchange sites per gram Membrane (×10⁻⁴ mol SO₃/g)Chitosan 10.5 ± 0.8 Butyl Modified 226 ± 21 Hexyl Modified 167 ± 45Octyl Modified  529 ± 127 Decyl Modified  483 ± 110Further, such polycationic polymers are capable of immobilizing enzymesand increasing the activity of enzymes immobilized therein as comparedto the activity of the same enzyme in a buffer solution. In variousembodiments, the polycationic polymers are hydrophobically modifiedpolysaccharides, particularly, hydrophobically modified chitosan. Forexample, for the hydrophobic modifications noted, the enzyme activitiesfor glucose oxidase were measured. The highest enzyme activity wasobserved for glucose oxidase in a hexyl modified chitosan suspended int-amyl alcohol. These immobilization membranes showed a 2.53 foldincrease in glucose oxidase enzyme activity over enzyme in buffer. Table2 details the glucose oxidase activities for a variety ofhydrophobically modified chitosans.

TABLE 2 Glucose oxidase enzyme activity for modified chitosans EnzymeActivity Membrane/Solvent (Units/gm) Buffer 103.61 ± 3.15 UNMODIFIEDCHITOSAN  214.86 ± 10.23 HEXYL CHITOSAN Chloroform  248.05 ± 12.62t-amyl alcohol 263.05 ± 7.54 50% acetic acid 118.98 ± 6.28 DECYLCHITOSAN Chloroform  237.05 ± 12.31 t-amyl alcohol  238.05 ± 10.02 50%acetic acid  3.26 ± 2.82 OCTYL CHITOSAN Chloroform 232.93 ± 7.22 t-amylalcohol 245.75 ± 9.77 50% acetic acid  127.55 ± 11.98 BUTYL CHITOSANChloroform 219.15 ± 9.58 t-amyl alcohol 217.10 ± 6.55 50% acetic acid127.65 ± 3.02

To prepare the hydrophobically modified chitosans of the inventionhaving an alkyl group as a modifier, a chitosan gel was suspended inacetic acid followed by addition of an alcohol solvent. To this chitosangel was added an aldehyde (e.g., butanal, hexanal, octanal, or decanal),followed by addition of sodium cyanoborohydride. The resulting productwas separated by vacuum filtration and washed with an alcohol solvent.The modified chitosan was then dried in a vacuum oven at 40° C. andresulted in a flaky white solid.

To prepare a hydrophobically modified chitosan of the invention having aredox mediator as a modifier, a redox mediator ligand was derivatized bycontacting 4,4′-dimethyl-2,2′-bipyridine with lithium diisopropylaminefollowed by addition of a dihaloalkane to produce4-methyl-4′-(6-haloalkyl)-2,2′-bipyridine. This ligand was thencontacted with Ru(bipyridine)₂Cl₂ hydrate in the presence of aninorganic base and refluxed in a water-alcohol mixture until theRu(bipyridine)₂Cl₂ was depleted. The product was then precipitated withammonium hexafluorophosphate, or optionally a sodium or potassiumperchlorate salt, followed by recrystallization. The derivatized redoxmediator (Ru(bipyridine)₂(4-methyl-4′-(6-bromohexyl)-2,2′-bipyridine)⁺²)was then contacted with deacetylated chitosan and heated. The redoxmediator modified chitosan was then precipitated and recrystallized.

The hydrophobically modified chitosan membranes have advantageousinsolubility in ethanol. For example, the chitosan enzyme immobilizationmaterials described above generally are functional to immobilize andstabilize the enzymes in solutions having up to greater than about 99wt. % or 99 volume % ethanol. In various embodiments, the chitosanimmobilization material is functional in solutions having 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more wt. % orvolume % ethanol. In some instances the chitosan immobilization materialis functional in solutions having from about 15 to about 95 wt. % orvol. % ethanol, from about 25 to about 95 wt. % or vol. % ethanol, fromabout 35 to about 95 wt. % or vol. % ethanol, from about 45 to about 95wt. % or vol. % ethanol, from about 55 to about 95 wt. % or vol. %ethanol, from about 65 to about 95 wt. % or vol. % ethanol, from about70 to about 95 wt. % or vol. % ethanol, from about 75 to about 95 wt. %or vol. % ethanol, from about 80 to about 95 wt. % or vol. % ethanol,from about 85 to about 95 wt. % or vol. % ethanol, or from about 90 toabout 95 wt. % or vol. % ethanol.

In other embodiments, the micellar or inverted micellar immobilizationmaterials are polyanionic polymers, such as hydrophobically modifiedpolysaccharides, particularly, hydrophobically modified alginate.Alginates are linear unbranched polymers containing β-(1-4)-linkedD-mannuronic acid and α-(1-4)-linked L-guluronic acid residues. In theunprotonated form, β-(1-4)-linked D-mannuronic acid corresponds to thestructure of Formula 3A

and in the unprotonated form, α-(1-4)-linked L-guluronic acidcorresponds to the structure of Formula 3B(Note structures 3a and 3Bcould be made better by showing bonding to the C6 carboxylate to thecarbon and, in 3A, bonding of C3 to the oxygen in the hydroxyl group.)

Alginate is a heterogeneous polymer consisting of polymer blocks ofmannuronic acid residues and polymer blocks of guluronic acid residues.

Alginate polymers can be modified in various ways. One type is alginatemodified with a hydrophobic cation that is larger than the ammonium (NH₄⁺) ion. The hydrophobic cation serves the dual function of (1) dictatingthe polymer's pore size and (2) acting as a chemical buffer to helpmaintain the micelle's pH level, both of which stabilize the enzyme.With regard to the first function of the hydrophobic cation, modifyingalginate with a hydrophobic cation produces an immobilization materialwherein the micelle size is dependent on the size of the hydrophobiccation. Accordingly, the size, shape, and extent of the modification ofthe alginate with the hydrophobic cation affects the size and shape ofthe micellar pore/pocket. This function of the hydrophobic cation allowsthe micelle size to be made larger or smaller or a different shape tofit a specific enzyme by varying the size and branching of thehydrophobic cation.

Regarding the second function of the hydrophobic cation, the propertiesof the alginate polymer are altered by exchanging the hydrophobic cationfor protons as the counterion to the —CO₂ ⁻ groups on the alginate. Thischange in counterion provides a buffering effect on the pH because thehydrophobic cation has a much greater affinity for the —CO₂ ⁻ sites thanprotons do. This buffering effect of the alginate membrane causes the pHof the micellar pore/pocket to remain substantially unchanged withchanging solution pH; stated another way, the pH of the pore resistschanges in the solution's pH. In addition, the alginate membraneprovides a mechanical barrier, which further protects the immobilizedenzymes.

In order to prepare a modified alginate membrane, the first step is tocast a suspension of alginate polymer with a solution of the hydrophobiccation to form a membrane. The excess hydrophobic cations and theirsalts are then extracted from the membrane, and the membrane is re-cast.Upon re-casting, the membrane contains the hydrophobic cations inassociation with —CO₂ ⁻ sites of the alginate membrane. Removal of thesalts of the hydrophobic cation from the membrane results in a morestable and reproducible membrane; if they are not removed, the excesssalts can become trapped in the pore or cause voids in the membrane.

In one embodiment, a modified alginate membrane is prepared by casting asuspension of alginate polymer with a solution of a salt of ahydrophobic cation such as quaternary ammonium bromide. Excessquaternary ammonium bromide or hydrogen bromide is removed from themembrane before it is re-cast to form the salt-extracted membrane. Saltextraction of membranes retains the presence of the quaternary ammoniumcations at the carboxylic acid exchange sites, but eliminatescomplications from excess salt that may be trapped in the pore or maycause voids in the equilibrated membrane. Exemplary hydrophobic cationsare ammonium-based cations, quaternary ammonium cations,alkyltrimethylammonium cations, alkyltriethylammonium cations, organiccations, phosphonium cations, triphenylphosphonium, pyridinium cations,imidazolium cations, hexadecylpyridinium, ethidium, viologens, methylviologen, benzyl viologen, bis(triphenylphosphine)iminium, metalcomplexes, bipyridyl metal complexes, phenanthroline-based metalcomplexes, [Ru(bipyridine)₃]²⁺ and [Fe(phenanthroline)₃]³⁺.

In one preferred embodiment, the hydrophobic cations are ammonium-basedcations. In particular, the hydrophobic cations are quaternary ammoniumcations. In another embodiment, the quaternary ammonium cations arerepresented by Formula 4:

wherein R₁, R₂, R₃, and R₄ are independently hydrogen, hydrocarbyl,substituted hydrocarbyl, or heterocyclo wherein at least one of R₁, R₂,R₃, and R₄ is other than hydrogen. In a further embodiment, preferably,R₁, R₂, R₃, and R₄ are independently hydrogen, methyl, ethyl, propyl,butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl,tridecyl or tetradecyl wherein at least one of R₁, R₂, R₃, and R₄ isother than hydrogen. In still another embodiment, R₁, R₂, R₃, and R₄ arethe same and are methyl, ethyl, propyl, butyl, pentyl or hexyl. In yetanother embodiment, preferably, R₁, R₂, R₃, and R₄ are butyl. In yetanother embodiment, preferably, R₁, R₂, R₃, and R₄ are ethyl.Preferably, the quaternary ammonium cation is tetraethylammonium,tetrapropylammonium (T3A), tetrapentylammonium (T5A), tetrahexylammonium(T6A), tetraheptylammonium (T7A), trimethylicosylammonium (TMICA),trimethyloctyldecylammonium (TMODA), trimethylhexyldecylammonium(TMHDA), trimethyltetradecylammonium (TMTDA), trimethyloctylammonium(TMOA), trimethyldodecylammonium (TMDDA), trimethyldecylammonium (TMDA),trimethylhexylammonium (TMHA), tetrabutylammonium (TBA),triethylhexylammonium (TEHA), and combinations thereof.

The micelle characteristics were studied and the micellar pore/pocketstructure of this membrane is ideal for enzyme immobilization, becausethe micellar pores/pockets are hydrophobic, micellar in structure,buffered to external pH change, and have high pore interconnectivity.

In another experiment, ultralow molecular weight alginate anddodecylamine were placed in 25% ethanol and refluxed to produce adodecyl-modified alginate by amidation of the carboxylic acid groups.Various alkyl amines can be substituted for the dodecylamine to producealkyl-modified alginate having a C₄-C₁₆ alkyl group attached to varyingnumbers of the reactive carboxylic acid groups of the alginatestructure. In various embodiments, at least about 1, 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48%,or more of the carboxylic acid groups react with the alkylamine. In someinstances, from about 2 to about 50%, from about 10 to about 50%, fromabout 20 to about 50%, from about 30 to about 50%, from about 40 toabout 50% of the carboxylic acid groups react with the alkylamine.

The hydrophobically modified alginate membranes have advantageousinsolubility in ethanol. For example, the alginate enzyme immobilizationmaterials described above generally are functional to immobilize andstabilize the enzymes in solutions having at least about 25 wt. % or 25volume % ethanol. In various embodiments, the alginate immobilizationmaterial is functional in solutions having 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90 or more wt. % or volume % ethanol. In someinstances, the alginate immobilization material is functional insolutions having from about 25 to about 95 wt. % or vol. % ethanol, fromabout 35 to about 95 wt. % or vol. % ethanol, from about 45 to about 95wt. % or vol. % ethanol, from about 55 to about 95 wt. % or vol. %ethanol, from about 65 to about 95 wt. % or vol. % ethanol, from about70 to about 95 wt. % or vol. % ethanol, from about 75 to about 95 wt. %or vol. % ethanol, from about 80 to about 95 wt. % or vol. % ethanol,from about 85

In order to evaluate the most advantageous immobilization material for aparticular enzyme, the selected enzyme can be immobilized in variousimmobilization materials, deposited on an electron conductor, andtreated with a solution containing an electron mediator (e.g., NAD⁺)and/or a substrate for the particular enzyme in a buffer solution. Afluorescence micrograph is obtained and shows fluorescence when theenzyme immobilized in the particular immobilization material is still acatalytically active enzyme after immobilization. Enzyme activity couldalso be determined by any standard spectroscopic assay. Further, enzymeactivity can be determined using a bioreactor for that enzyme, inparticular, the activity of carbonic anhydrase can be measured by usingthe bioreactor described in example 1 or a carbonic anhydrase assay aspublished by Sigma (revision date Jul. 22, 1996). The carbonic anhydraseassay measures the rate of enzymatic CO₂ hydration by determining thenet rate difference between a non-enzymatic blank and anenzyme-containing sample in the time required to decrease the pH of abuffered reaction mixture from 8.3 to 6.3.

The assay techniques described above are one way to determine whether aparticular immobilization material will immobilize and stabilize anenzyme while retaining the enzyme's catalytic activity. For example, forstarch-consuming amylase, the enzyme immobilization material thatprovided the greatest relative activity is provided by immobilization ofthe enzyme in butyl chitosan suspended in t-amyl alcohol. Formaltose-consuming amylase, the greatest relative activity is provided byimmobilization of the enzyme in medium molecular weight decyl modifiedchitosan.

One aspect of the present invention is directed to an enzyme immobilizedby entrapment in a polymeric immobilization material, the immobilizationmaterial being permeable to a compound smaller than the enzyme andhaving the structure of either Formulae 5, 6, 7, or 8:

wherein R₂₁, R₂₂, R₂₃ and R₂₄ are independently hydrogen, alkyl, orsubstituted alkyl, provided that the average number of alkyl orsubstituted alkyl groups per repeat unit is at least 0.1 R₂₅ is hydrogenor substituted alkyl, provided that the average number of substitutedalkyl groups per repeat unit is at least 0.1; R₃₂ and R₃₃ areindependently hydrogen, alkyl, aryl, or substituted alkyl, provided thatthe average number of hydrogen atoms per repeat unit is at least 0.1 andm, n, o, and p are independently integers of from about 10 to about5000. In many of these embodiments, the enzyme immobilization materialcomprises a micellar or inverted micellar polymer.

Modified Polysulfone

In some of the various embodiments, the immobilization material has astructure of Formula 5

wherein R₂₁, R₂₂, and n are defined above. In various embodiments, R₂₁and R₂₂ are independently hydrogen, alkyl, or substituted alkyl. Invarious embodiments, R₂₁ and R₂₂ are independently hydrogen or—(CH₂)_(q)N⁺R₂₆R₂₇R₂₈, wherein R₂₆, R₂₇, and R₂₈ are independently alkyland q is an integer of 1, 2, or 3; particularly, R₂₆, R₂₇, and R₂₈ areindependently methyl, ethyl, propyl, butyl, pentyl, or hexyl; moreparticularly, R₂₆, R₂₇, and R₂₈ are methyl.

Alternatively, R₂₁ and R₂₂ are independently hydrogen or—(CH₂)_(q)N⁺R₂₆R₂₇R₂₈, wherein R₂₆ and R₂₇ are independently methyl,ethyl, or propyl, R₂₈ is alkylamino, and q is an integer of 1, 2, or 3.When R₂₈ is alkylamino, preferred alkylamino groups are tertiaryalkylamino groups. For example, the alkylamino group can be—CH₂N⁺R₂₉R₃₀R₃₁, —CH₂CH₂N⁺R₂₉R₃₀R₃₁ or —CH₂CH₂CH₂N⁺R₂₉R₃₀R₃₁ whereinR₂₉, R₃₀, and R₃₁ are independently hydrogen or alkyl. In variouspreferred embodiments, R₂₉, R₃₀, and R₃₁ are independently methyl,ethyl, propyl, butyl, pentyl, or hexyl; more particularly, R₂₉, R₃₀, andR₃₁ are methyl or ethyl.

Preferably, R₂₁, R₂₂, or R₂₁ and R₂₂ are alkyl or substituted alkylwherein the average number of alkyl or substituted alkyl groups perrepeat unit is from about 0.1 to about 1.4, from about 0.2 to about 1.4,from about 0.3 to about 1.4, from about 0.3 to about 1.2, from about 0.3to about 1, from about 0.3 to about 0.8, from about 0.4 to about 1.4,from about 0.4 to about 1.2, from about 0.4 to about 1, from about 0.4to about 0.8, from about 0.5 to about 1.4, from about 0.5 to about 1.2,from about 0.5 to about 1, from about 0.5 to about 0.8.

In other preferred embodiments, R₂₁ and R₂₂ are independently hydrogenor —(CH₂)_(q)-polyether wherein q is an integer of 1, 2, or 3. Inpreferred embodiments, q is 1. In some of the preferred embodiments, R₂₁and R₂₂ are independently hydrogen, —CH₂—O —(CH₂(CH₃)—CH₂—O)_(z)—R_(t),—CH₂—O—(CH₂—CH₂—O)_(z)—R_(t), or a combination thereof wherein z is aninteger from 3 to 180, and the polyethylene oxide or polypropylene oxide(e.g., —O —(CH₂—CH₂—O)_(z)—R_(t) or —CH₂—O—(CH₂(CH₃)—CH₂—O)_(z)—R_(t)wherein R_(t) is hydrogen, alkyl, substituted alkyl, aryl, orsubstituted aryl) has a molecular weight from about 150 Daltons (Da) toabout 8000 Daltons (Da). In particular embodiments, the polyethyleneoxide has a molecular weight from about 500 Da to about 600 Da;particularly about 550 Da.

Modified polysulfone is a desirable immobilization material because ithas good chemical and thermal stability. Additionally, modifiedpolysulfone has advantageous solubility characteristics in polar organicsolvents such as N-methylpyrrolidone (NMP) and dioxane. This solubilityenables the modified polysulfone beads to be prepared by precipitationin water or lower aliphatic alcohols. Unmodified polysulfone canimmobilize and retain an enzyme (e.g., carbonic anhydrase) in the beads.But, the activity of the carbonic anhydrase is reduced and it ishypothesized that the low porosity and thus, the low permeability ofunmodified polysulfone beads at the polymer-solvent interface preventsthe substrate and product from diffusing to and from the active site ofthe enzyme. In order to improve the porosity, the polysulfone can bemodified to increase the porosity and transport of the substrate andproduct through the material.

For example, the polysulfone can be modified by adding amine groups tothe benzene groups of the polysulfone. By modifying the polysulfone withquaternary amine groups, the hydrophilicity of the polysulfone isaffected and in turn the porosity and the transport ofcarbonate/bicarbonate ions increases. Also, the positively charged aminegroups can stabilize carbonic anhydrase through electrostaticinteractions. This modification of adding a hydrophobic group to ahydrophilic polymer may also form micellar aggregate/pore structures inthe polymer. To add amine groups to the polysulfone, the benzene ringsof the backbone are chloromethylated followed by the amination of thechloromethyl groups. This process is generally described in Jihua, H.;Wentong, W.; Puchen, Y.; Qingshuang, Z. Desalination 1991, 83, 361 andPark, J.-S.; Park, G.-G.; Park, S.-H.; Yoon, Y.-G.; Kim, C. S.; Lee, W.Y. Macromol. Symp. 2007, 249-250, 174. The general reaction scheme forthis transformation is shown in Scheme 1. The average number ofchloromethyl groups added per repeat unit can be controlled bymanipulating the reactant ratios during the first step as described inHibbs, M. R.; Hickner, M. A.; Alam, T. M.; McIntyre, S. K.; Fujimoto, C.H.; Cornelius, C. J. Chem. Mater. 2008, 20, 2566.

Additionally, the choice of tertiary amine added to the chloromethylatedpolysulfone (PSf-CH₂Cl) can affect the polysulfone properties. Forinstance, trimethyl amine can be used to aminate PSf-CH₂Cl, resulting ina quaternary benzyl trimethyl ammonium cation. This benzyl trimethylammonium cation has been shown to be more stable with prolonged exposureto elevated temperatures and/or strongly basic solutions. (See Sata, T.;Tsujimoto, M.; Yamaguchi, T.; Matsusaki, K. J. Membrane Sci. 1996, 112,161.) Tertiary diamines can also be used in this amination step,providing a way of crosslinking polysulfone to improve its mechanicaland thermal stability. The addition of diamines to chloromethylatedpolysulfone solutions crosslinks polysulfone and solidifies the mixture.The solvent can then be exchanged with water or methanol to yield a moreporous aminated polysulfone. The initial polymer concentration of thesolution can be adjusted to manipulate the porosity in the resultingpolysulfone. The exchange of the chloride anions with bicarbonate anionsafter amination could improve the performance of the immobilizedcarbonic anhydrase by removing chloride ions that inhibit enzymeactivity. Additionally, the incorporation of bicarbonate ions intopolysulfone could provide a buffering capacity to protect the enzymefrom pH changes.

Further, once the polysulfone is chloromethylated, other modifiedpolysulfone polymers can be prepared. For example, the chloromethylgroups can react with a hydroxyl end group of poly(ethylene oxide) (PEO)to create polysulfone polymers with grafted PEO side chains. (See Park,J. Y.; Acar, M. H.; Akthakul, A.; Kuhlman, W.; Mayes, A. M. Biomater.2006, 27, 856.) The general reaction scheme is shown in Scheme 2. Asdescribed above, the chloromethylation of polysulfone can be manipulatedto provide control over the grafting density of the PEO side chains.Additionally, the molecular weight of the PEO side chains can be alteredto influence the overall weight loading of PEO in PEO-modifiedpolysulfone; the loading affects the overall mechanical properties ofthe polymer.

The incorporation of PEO into polysulfone will improve thehydrophilicity of these beads and the transport of carbonate/bicarbonateions. Additionally, when polyethylene glycol-modified carbonic anhydraseis the enzyme, the PEO-modified polysulfone can provide a hydrophilicPEO layer around the carbonic anhydrase and further prevent the enzymefrom leaching. The PEO encapsulation of carbonic anhydrase can alsoprotect the enzyme from effects of drying that may be important forretaining its activity upon immobilization.

Additionally, particular processing conditions can also improve theporosity and the ion transport of the polymers. For instance, it ispossible to foam polysulfone through the use of supercritical carbondioxide to introduce microporous structure into polysulfone polymers.(See Krause, B.; Mettinkhof, R.; van der Vegt, N. F. A.; Wessling, M.Macromolecules 2001, 34, 874.) A similar approach could be used toenable the foaming of modified polysulfone beads. Microporosity can alsobe introduced into polysulfone by using a freeze-drying process similarto the process used to create microporous chitosan. (See Cooney, M. J.;Lau, C.; Windmeisser, M.; Liaw, B. Y.; Klotzbach, T.; Minteer, S. D. J.Mater. Chem. 2008, 18, 667.) Since polysulfone is not soluble in awater/acetic acid mixture, a suitable solvent for polysulfone that iscapable of appreciable sublimation in its solid state under vacuum isrequired. Menthol is a promising candidate due to its low meltingtemperature (35° C.) and comparable solubility parameter to dioxane,which suggests that polysulfone could dissolve at high concentrations inmenthol at slightly elevated temperatures.

Modified Polycarbonate

In certain embodiments, the immobilization material has a structure ofFormula 6

wherein R₂₃, R₂₄, and m are defined above. In various embodiments, R₂₃and R₂₄ are independently hydrogen, alkyl, or substituted alkyl. Invarious embodiments, R₂₃ and R₂₄ are independently hydrogen or—(CH₂)_(q)N⁺R₂₆R₂₇R₂₈, wherein R₂₆, R₂₇, and R₂₈ are independently alkyland q is an integer of 1, 2, or 3; particularly, R₂₆, R₂₇, and R₂₈ areindependently methyl, ethyl, propyl, butyl, pentyl, or hexyl; moreparticularly, R₂₆, R₂₇, and R₂₈ are methyl.

Alternatively, R₂₃ and R₂₄ are independently hydrogen or—(CH₂)_(p)N⁺R₂₆R₂₇R₂₈ wherein R₂₆ and R₂₇ are independently methyl,ethyl, or propyl, R₈ is alkylamino, and p is an integer of 1, 2, or 3.When R₂₈ is alkylamino, preferred alkylamino groups are tertiaryalkylamino groups. For example, the alkylamino group can be—CH₂N⁺R₂₉R₃₀R₃₁, —CH₂CH₂N⁺R₂₉R₃₀R₃₁ or —CH₂CH₂CH₂N⁺R₂₉R₃₀R₃₁ whereinR₂₉, R₃₀, and R₃₁ are independently hydrogen or alkyl. In variouspreferred embodiments, R₂₉, R₃₀, and R₃₁ are independently methyl,ethyl, propyl, butyl, pentyl, or hexyl; more particularly, R₂₉, R₃₀, andR₃₁ are methyl or ethyl.

In other preferred embodiments, R₂₃ and R₂₄ are independently hydrogenor —(CH₂)_(q)-polyether wherein q is an integer of 1, 2, or 3. In someof the preferred embodiments, R₂₃ and R₂₄ are independently hydrogen,—CH₂—O—(CH₂(CH₃)—CH₂—O)_(z)—R_(t), —CH₂—O—(CH₂—CH₂—O)_(z)—R_(t), or acombination thereof wherein z is an integer from 3 to 180, and thepolyethylene oxide or polypropylene oxide (e.g.,—O—(CH₂—CH₂—O)_(z)—R_(t) or —CH₂—O—(CH₂(CH₃)—CH₂—O)_(z)—R_(t) whereinR_(t) is hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl)has a molecular weight from about 150 Daltons (Da) to about 8000 Daltons(Da).

Preferably, R₂₃, R₂₄, or R₂₃ and R₂₄ are alkyl or substituted alkylwherein the average number of alkyl or substituted alkyl groups perrepeat unit is from about 0.1 to about 1.4, from about 0.2 to about 1.4,from about 0.3 to about 1.4, from about 0.3 to about 1.2, from about 0.3to about 1, from about 0.3 to about 0.8, from about 0.4 to about 1.4,from about 0.4 to about 1.2, from about 0.4 to about 1, from about 0.4to about 0.8, from about 0.5 to about 1.4, from about 0.5 to about 1.2,from about 0.5 to about 1, from about 0.5 to about 0.8.

Polycarbonate has a structure similar to polysulfone. It also containsbenzene rings in its backbone, so it can be functionalized by addingchloromethyl groups in the same manner as described above forpolysulfone. These chloromethyl groups can then be aminated or have PEOgrafted following the same procedure utilized for polysulfone. Schemes 3and 4 show the general reaction schemes for both. Similar topolysulfone, polycarbonate can be foamed using supercritical carbondioxide.

Modified Poly(vinylbenzyl chloride)

In other embodiments, the immobilization material has a structure ofFormula 7

wherein R₂₅ and o are defined above. In various embodiments, R₂₅ ishydrogen, alkyl, or substituted alkyl. In various embodiments, R₂₅ ishydrogen or —(CH₂)_(q)N⁺R₂₆R₂₇R₂₈, wherein R₂₆, R₂₇, and R₂₈ areindependently alkyl and q is an integer of 1, 2, or 3; particularly,R₂₆, R₂₇, and R₂₈ are independently methyl, ethyl, propyl, butyl,pentyl, or hexyl; more particularly, R₂₆, R₂₇, and R₂₈ are methyl.

Alternatively, R₂₅ is hydrogen or —(CH₂)_(p)N⁺R₂₆R₂₇R₂₈ wherein R₂₆ andR₂₇ are independently methyl, ethyl, or propyl, R₂₈ is alkylamino, and pis an integer of 1, 2, or 3. When R₂₈ is alkylamino, preferredalkylamino groups are tertiary alkylamino groups. For example, preferredalkylamino groups can be —CH₂N⁺R₂₉R₃₀R₃₁, —CH₂CH₂N⁺R₂₉R₃₀R₃₁ or—C₆H₄N⁺R₂₉R₃₀R₃₁ wherein R₂₉, R₃₀, and R₃₁ are independently hydrogen oralkyl. In various preferred embodiments, R₂₉, R₃₀, and R₃₁ areindependently methyl, ethyl, propyl, butyl, pentyl, or hexyl; moreparticularly, R₂₉, R₃₀, and R₃₁ are methyl or ethyl.

Preferably, R₂₅ is substituted alkyl wherein the average number ofsubstituted alkyl groups per repeat group is at least 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, or more.

Poly(vinylbenzyl chloride) (PVBC) is a commercially-available polymerwith a chloromethyl group contained in the polymer, so it can beaminated similarly to the synthetic procedure described above forchloromethylated polysulfone or polycarbonate. PVBC, however, lacks themechanical strength of polysulfone and polycarbonate and is somewhatbrittle and has a lower glass transition temperature. However, it isbelieved that the mechanical and thermal stability of this polymer canbe improved by crosslinking PVBC by amination with tertiary diamines.(See Varcoe, J. R.; Slade, R. C. T.; Lee, E. L. H. Chem. Commun. 2006,1428.) This process incorporates positive charges in the PVBC and thesecharges can also stabilize the immobilized enzyme through electrostaticinteractions. Scheme 5 shows the general scheme for this reaction.

Upon addition of a diamine to a 40 wt. % solution of PVBC in NMP, both amethylene (—CH₂—) and a phenylene (—C₆H₄—) spacer in the diamineproduces crosslinked solid films. Diamines having the followingstructures were selected because they provide long-term stability tothese quaternary amines. The use of tetramethyl methanediamine (TMMDA)solidifies this solution quickly (e.g., less than 10 minutes),indicating that the reaction of TMMDA with PVBC is fast. Oncesolidified, PVBC crosslinked with TMMDA does not swell upon addition ofmethanol or water. In contrast, the reaction of tetramethylphenylenediamine (TMPDA) is slower and takes several hours to solidify.Once solidified, PVBC crosslinked with TMPDA swells significantly (butmaintains its original shape) upon exposure to either methanol or water.PVBC crosslinked with TMPDA forms a hydrophilic, high-swelling material,which could significantly improve the transport of carbonate/bicarbonateions through the polymer, as compared to polysulfone and polycarbonatethat are rigid glassy polymers. Similar to the polysulfone andpolycarbonate, the amount of derivatization of the modified PVBC can bealtered by adjusting the polymer concentration of the solution duringthe chloromethylation reaction.

Modified Polysiloxanes

In various embodiments, the immobilization material has a structure ofFormula 8

wherein R₃₂ and R₃₃ are independently hydrogen, alkyl, aryl, orsubstituted alkyl, provided that the average number of hydrogen atomsper repeat unit is at least 0.1.

In various embodiments, R₃₂ and R₃₃ are independently hydrogen, alkyl,aryl, -(substituted alkylene)-acid or a salt thereof, -(substitutedalkylene)-base or a salt thereof, —(CH₂)_(q)O—(CH₂—CH₂—O)_(z)—R_(t),—CH₂—O—(CH₂(CH₃)—CH₂—O)_(z)—R_(t), or a combination thereof, wherein qis an integer of 2, 3, or 4 and R_(t) is. The acid group can be acarboxylic, a phosphonic, a phosphoric, a sulfonic, a sulfuric, asulfamate, a salt thereof, or a combination thereof. The base can be anamine base, particularly, a tertiary amine, a quaternary amine, anitrogen heterocycle, a salt thereof, or a combination thereof. Inparticular embodiments, R₃₂ and R₃₃ are independently hydrogen, alkyl,aryl, —(CH₂)₃—O—((CH₂)₂—O—)_(z)CH₃, —(CH₂)₂—C(O)—O—(CH₂)₂-imidazolium,—(CH₂)₃—O—CH₂—CH(OH)—N(CH₃)—(CH₂)₂—SO₃Na.

The structure of Formula 8 is prepared starting with a hydrosiloxane,which is a polysiloxane that contains silicon hydride bonds. Examplesinclude poly(methyl hydrosiloxane) (PMHS) homopolymer, poly(phenyldimethylhydrosiloxy)siloxane (PPDMHS) homopolymer, and copolymers ofPMHS or PPDMHS with other polysiloxanes such as poly(dimethylsiloxane)(PDMS) or poly(phenylmethylsiloxane) (PPMS). Specifically, polyalkylhydrosiloxane (e.g., poly(methyl hydrosiloxane), poly(ethylhydrosiloxane), poly(propyl hydrosiloxane), polyaryl hydrosiloxane(e.g., poly(phenyl hydrosiloxane), poly(tolyl hydrosiloxane)),poly(phenyl dimethylhydrosiloxy)siloxane, poly(dimethyl siloxaneco-methyl hydrosiloxane), poly(methyl hydrosiloxane co-phenyl methylsiloxane), poly(methyl hydrosiloxane co-alkyl methyl siloxane),poly(methyl hydrosiloxane co-diphenyl siloxane), poly(methylhydrosiloxane co-phenyl methyl siloxane). These polysiloxanes have adesirable CO₂ solubility. Without being bound by theory, it is believedthat the elasticity of polysiloxanes increases CO₂ solubility. Usingpublished procedures, these hydride-functional polysiloxanes can begrafted with polyether and/or ionic groups by coupling them withallyl-containing compounds using a platinum catalyst (hydrosilationreaction). The general reaction schemes are shown in Schemes 6-8.

Generally, functionalization of an ionic and nonionic polysiloxane canbe manipulated by controlling the amount of polyether or ionic groupsadded. In particular, functionalization of PMHS can be varied by varyingthe amount of allyl PEG, allyl glycidyl ether, and/or alkylimidazoliumacrylate added to the reaction mixture. Addition of functional sites(e.g., polyether or ionic groups) increases the water solubility ofionic and nonionic polysiloxanes. The water solubility of the polymerdepends on the number of functional sites added to the polysiloxane.Further, polysiloxanes can be functionalized with both a polyether andan ionic species by adding a polyether having an allyl group and anionic compound having an allyl group to the same reaction mixture.

The functionalized PMHS can then be crosslinked into an elastomer havingproperties similar to a natural rubber by using the remaining Si—Hgroups via two possible pathways, a hydrosilylation reaction or adehydrogenative coupling reaction. The hydrosilylation reaction uses aplatinum catalyst such as platinum-divinyltetramethyldisiloxane complexand vinyl-functional polysiloxanes as crosslinkers. Examples ofvinyl-functional polysiloxanes include divinyl-terminated PDMS or PPMS,poly(vinylmethylsiloxane) (PVMS) homopolymer, and copolymers of PVMS andPDMS or PPMS. The dehydrogenative coupling reaction uses a catalystwherein the choice of catalyst depends on the coupling mechanism. Tincatalysts are predominately used in dehydrogenative coupling reactionwhere Si—H couples to Si—OH to form Si—O—Si linkages. Tin catalyst suchas di-n-butyldilauryltin are used with silanol-functional polysiloxanesas crosslinkers. In addition to tin compounds, other transition metalcomplexes based on zinc, iron, cobalt, ruthenium, iron, rhodium,iridium, palladium, and platinum can be used. Specific examples includezinc octoate, iron octoate, and Wilkinson's catalyst (rhodium-basedmetal salt; (PhP)₃RhCl). Precious metal catalysts (predominatelyplatinum but rhodium as well) are used in hydrosilylation reactionswhere Si—H reacts with a terminal vinyl bond to form Si—CH₂—CH₂—Si. Freeradical initiators (thermal and/or UV generated) can be used tocrosslink vinyl, acrylate, or methacrylate containing polysiloxanes. Tinand/or titanium compounds are used to catalyze condensation cure systemswhere Si—OH groups react with a variety of reactive groups (alkoxy,acetoxy, oxime, enoxy, and amines) to form Si—O—Si bonds. Thesecondensation cure systems are moisture sensitive and will react in thepresence of water only, but using titanium and/or tin compounds speedsup that reaction. Examples of silanol-functional polysiloxanes includedisilanol-terminated PDMS or poly(trifluoropropylmethylsiloxane)(PTFPMS), disilanol-terminated copolymers of PPMS and PDMS, andsilanol-trimethylsilyl modified Q resins. The crosslink density affectsthe material's properties and enzyme retention in the immobilizationmatrix.

Other variables to this immobilization procedure include annealingtemperature (4° C.-60° C. for bovine carbonic anhydrase (BCA) or to 80°C. for human carbonic anhydrase (HCA)) and tin catalyst choice andloading. In addition, to dibutyldilauryltin, bis(2-ethylhexanoate)tin,dimethylhydroxy(oleate)tin, and dioctyldilauryltin can be used as thecatalyst. As the annealing temperature increases, the amount of tincatalyst needed to maintain a fast reaction rate (solidifying in 30minutes or less) decreases and ranges from about 0.01 to about 10 vol.%, preferably about 0.2 to about 4 vol. %.

The final geometry of the polymer pieces can be varied as well. Thecylinder diameter and length can be changed using different acrylicmolds. Alternatively, the polysiloxane can be coated onto a solidsupport. Ideally, the surface of the solid support will befunctionalized with Si—OH groups so that it can covalently bind to thepolysiloxane during the tin-catalyzed crosslinking reaction. The typeand molecular weight of the disilanol-terminated polymer crosslinker canalso be varied to change the composite polysiloxane properties (e.g.,density, mechanical strength, etc.). Alternatives to PDMS-(OH)₂ are thedisilanol terminated diphenylsiloxane-dimethylsiloxane copolymer,disilanol terminated poly(trifluoropropylmethylsiloxane), and disilanolterminated poly(diphenylsiloxane).

Additionally, PMHS-g-PEG can be crosslinked via a different mechanism(hydrosilylation) using precious metal catalysts and vinyl-containingpolysiloxane crosslinkers of various molecular weights. Useful catalystsfor this reaction are platinum-divinyltetramethyldisiloxane complex,platinum-cyclovinylmethylsiloxane complex, andtris(dibutylsulfide)rhodium trichloride at loadings of about 0.01 toabout 5 vol. %, preferably about 0.02 to about 0.5 vol. %. Examples ofvinyl-containing polysiloxane crosslinkers are divinyl terminatedpoly(dimethylsiloxane), divinyl terminateddiphenylsiloxane-dimethylsiloxane copolymer, divinyl terminatedpoly(phenylmethylsiloxane), poly(vinylmethylsiloxane), vinyl Q resins,vinyl T structure polymers, vinylmethylsiloxane-dimethylsiloxanecopolymer, and poly(vinylphenylsiloxane co-phenylmethylsiloxane).

Enzyme Encapsulation Process

In order to encapsulate an enzyme in polysulfone, the enzyme must not bedeactivated by the solvent used for dissolving the polymer. Whenpreparing the enzyme encapsulated polymer beads, the enzyme is dissolvedinto a solvent with a surfactant. Next the polysulfone is added to thesolution and stirred until completely dissolved. The polysulfone/enzymesolution is held at room temperature until complete mixing has beenachieved. The dissolved polysulfone/enzyme solution is then addeddropwise to a water, an alcohol, or a water-alcohol solution; thisprocess forms polymeric beads as shown in FIG. 1. Additionally, FIG. 1illustrates the retention of soluble species in the polymeric bead. Theblue bead was created by mixing in 20 mg/mL copper phthalocyanine intothe 20 wt. % polysulfone in 1-methylpyrrolidone solution. The solutionis then added dropwise into a beaker of water forming the beads. Thebeads are washed repeatedly with water, alcohol, and carbonate solutionto wash any free dye off the bead.

To immobilize an enzyme in alginate, a 2% unmodified alginate solutionis loaded with enzyme, and then added dropwise into a 2 wt. % calciumchloride solution and stirred for at least thirty minutes. The enzymeencapsulated alginate beads are then removed and gently dried with papertowels. Next, the beads are rolled into a 20 wt. % polysulfone solutionby hand to obtain a thin polysulfone film encapsulating the alginatebead. This film is needed because alginate beads dissolve in sodiumcarbonate solution without a polysulfone coating, but the enzyme isretained in the alginate when a thin film of polysulfone coats the bead.This procedure could be used for other polymers to control substratediffusion.

To immobilize an enzyme in poly(vinylbenzyl chloride) (PVBC), a PVBCsolution was made in a water displaceable solvent such as dioxane or1-methylpyrrolidone. Then carbonic anhydrase was added to the solutionand stirred until a uniform dissolution occurred. Once the solution isadequately dispersed, a diamine crosslinker is added and stirred untilthe solution is sufficiently viscous to form a bead. However, thestirring should not be long enough to allow the solution to gel. Whenthe viscosity is low enough to be easily pipetted using a transferpipette, the solution is added dropwise into a beaker of water andstirred to remove the excess solvent.

To immobilize an enzyme in polysulfone-graft-polyethylene glycol(PSf-g-PEG) polymers, dry PSf-g-PEG and a low boiling point solvent(e.g., dichloromethane, 1,2-dichloroethane, 1,4-dioxane, chloroform,tetrahydrofuran, toluene, 2-butanone, benzene, ethyl acetate,acetonitrile, acetone) is placed in a vessel until the PSf-g-PEGdissolves. A support material (e.g., porous lava rocks, porous silica,porous ceramics, porous polymeric beads or other appropriate support) isplaced in a beaker and enzyme (e.g., carbonic anhydrase) is added withstirring. Once homogenized, PSf-g-PEG is added and stirred to coat thesupport material. The contents are stirred continuously until thesolvent evaporates. The immobilized carbonic anhydrase-coated supportmaterial is placed in the vacuum oven to remove residual solvent andstored in bicarbonate solution.

Alternatively, enzyme can be immobilized in PSf-g-PEG by placing dryPSf-g-PEG in a vessel with a water-miscible solvent (e.g.,N-methylpyrrolidone, 1,4-dioxane, dimethyl sulfoxide, tetrahydrofuran,acetonitrile, acetone) and stirred until the PSf-g-PEG dissolves. Oncethe PSf-g-PEG is dissolved, enzyme (e.g., carbonic anhydrase) is addedand mixed thoroughly. The enzyme/PSf-g-PEG solution is added dropwise todeionized water to form polymer beads. The beads are stored inbicarbonate solution.

Core Component

The core is any particle that provides a support for the immobilizedenzyme layer and that can be spray-dried. The core particle can be, forexample, a polymer particle, a carbon particle, a zeolite particle, ametal particle, a ceramic particle, a metal oxide particle, a silicaparticle, or a combination thereof. In some embodiments, the coreparticle is an inert core particle. In various embodiments, the coreparticle is not a polymer particle. Preferred core particles do notadversely affect the stability of the enzyme or a chemicaltransformation involving the enzyme. For particular applications, thecore particles have an average diameter from about 200 nm to about 100μm, depending upon the intended use of the particles when coated withthe immobilized enzyme. For other applications, the core particles canhave dimensions appropriate to the system designed for the application.

Methods of Preparing Coated Particles

The coated particles are prepared by mixing a solution comprising anenzyme with a suspension comprising at least one core particle, animmobilization material, and a liquid medium and spray-drying theresulting mixture. The solution, suspension, and spray-drying step aredescribed in more detail below.

An enzyme solution comprising the enzyme and a solvent is used in thecoating procedure. The enzyme is combined with a solvent and mixed untila solution is formed. Acceptable enzymes are described in more detailabove. The solvent can be an aqueous solution, particularly a buffersolution, such as an acetate buffer or phosphate buffer. The buffer pHis designed to provide an acceptable pH for the particular enzyme to beimmobilized. Also, in various embodiments, the enzyme solution cancontain an electron mediator as described above.

A suspension is prepared by combining a core particle, the desiredimmobilization material and a liquid medium. Exemplary core particlesand immobilization materials are described above. The liquid medium canbe a solvent or buffer, such as an acetate buffer or phosphate buffer.When a buffer is used as the liquid medium, the buffer pH is selected toprovide an acceptable pH for the particular enzyme to be immobilized andcoated.

Once the enzyme solution and the suspension are prepared, they arecombined and mixed well. The resulting mixture is then dried. Apreferred drying method is spray-drying because the drying also resultsin coating of the core particles with the immobilized enzyme layer.Conventional spray drying techniques can be used in the methods of theinvention. Alternatives to spray-drying include other conventionalprocesses for forming coated particles, such as fluidized bedgranulation, spray dry granulation, rotogranulation, fluidized bed/spraydrying granulation, extrusion and spheronization.

In some of the various embodiments, the solution comprises from about0.1 wt. % to about 15 wt. % of the enzyme and about 85 wt. % to about99.1 wt. % of a solvent, and the suspension comprises from about 0.1 wt.% to about 50 wt. % of the core particles, from about 4 wt. % to about10 wt. % of the enzyme immobilization material, and from about 50 wt. %to about 75 wt. % of the liquid medium. Other ways to make the castingsolution include mixing the particles and the enzyme together in bufferto form a suspension and then adding solubilized immobilization materialto complete the mixture or by combining all of the materials at once toform a suspension.

In various preferred embodiments, a mixture of enzyme and enzymeimmobilization material can be coated onto supporting particles using aspray coating/drying technique. For example, an airbrush (e.g., PaascheVL series) can be used to generate an aerosol of the components of themixture and propel them towards a target. The aerosol is generated usingcompressed nitrogen gas regulated at about 25 psi. The mixture isairbrushed onto a surface such as a polycarbonate shield from a distanceof about 40 cm from the tip of the airbrush to the shield. The airbrushcan be moved in a raster pattern while moving vertically down thepolycarbonate target in a zigzag pattern applying the casting solution.This procedure is used to minimize the coating thickness on the shieldand minimize the particle-particle interaction while drying. The castingsolution is allowed to dry on the shield for about 20 minutes beforebeing collected by a large spatula/scraper.

For other particles that cannot be coated using the spray dryingtechnique described above, the particles can be coated by methods knownin the art such as dip coating, brush coating, spin coating, and thelike.

Support or Substrate

Once the enzyme has been immobilized within the enzyme immobilizationmaterial, this immobilized enzyme can be deposited on a support. Thesubstrate can be a material that provides the desired mechanical supportnecessary for the selected use. For example, the support may be afilter, a wire mesh, porous polymer, organic and inorganic membrane, andthe like when the immobilized enzyme is used as a catalyst for achemical transformation.

Aqueous Liquid

As described above, the aqueous liquid is contacted with theCO₂-containing gas to help absorb the CO₂ and increase the CO₂concentration in the aqueous liquid. In many preferred embodiments, theaqueous liquid comprises a base. The base is a proton acceptor. The baseis water soluble and does not denature the carbonic anhydrase. The basecan be a metal hydroxide, a quaternary ammonium hydroxide, a metalcarbonate, a quaternary ammonium carbonate, a quaternary ammoniumalkoxide, a metal amide, a metal alkyl, a metal alkoxide, metalsilanoate, an amine, an alkanolamine, a conjugate base of a weak acid,or a combination thereof. The metal hydroxides can include lithiumhydroxide, sodium hydroxide, potassium hydroxide, rubidium hydroxide,cesium hydroxide, magnesium hydroxide, calcium hydroxide, strontiumhydroxide, barium hydroxide, or a combination thereof. Also, ammoniumhydroxide can be used in the aqueous liquid. The metal carbonate can belithium carbonate, sodium carbonate, potassium carbonate, rubidiumcarbonate, cesium carbonate, magnesium carbonate, calcium carbonate,strontium carbonate, barium carbonate, ammonium carbonate, a carbonatesalt of an organic cation, or a combination thereof. For example, thecarbonate salt of an organic cation can be a tetraalkyleammoniumcarbonate (e.g., tetramethylammonium carbonate, tetraethylammoniumcarbonate, tetrapropylammonium carbonate, tetrabutylammonium carbonate,tetrapentylammonium carbonate, or tetrahexylammonium carbonate) analkyltrimethyl ammonium carbonate (e.g., ethyltrimethyl ammoniumcarbonate, propyltrimethyl ammonium carbonate, butyltrimethyl ammoniumcarbonate, pentyltrimethyl ammonium carbonate, hexyltrimethyl ammoniumcarbonate, hepyltrimethyl ammonium carbonate, octyltrimethyl ammoniumcarbonate, nonyltrimethyl ammonium carbonate, decyltrimethyl ammoniumcarbonate, dodecyltrimethyl ammonium carbonate, or undecyltrimethylammonium carbonate), an alkyltriethylammonium carbonate (e.g.,methyltriethyl ammonium carbonate, propyltriethyl ammonium carbonate,butyltriethyl ammonium carbonate, pentyltriethyl ammonium carbonate,hexyltriethyl ammonium carbonate, hepyltriethyl ammonium carbonate,octyltriethyl ammonium carbonate, nonyltriethyl ammonium carbonate,decyltriethyl ammonium carbonate, dodecyltriethyl ammonium carbonate, orundecyltriethyl ammonium carbonate), or a combination thereof.

The quaternary ammonium hydroxide, quaternary ammonium carbonate, orquaternary ammonium alkoxide can be benzyltrimethylammonium hydroxide,choline hydroxide, diethyldimethylammonium hydroxide,dimethyldodecylethylammonium hydroxide,N,N,N,N′,N′,N′-hexabutylhexamethylenediammonium dihydroxide,hexadecyltrimethylammonium hydroxide, hexamethonium hydroxide,triethylmethylammonium hydroxide, tributylmethylammonium hydroxide,trihexyltetradecylammonium hydroxide, tetrapropylammonium hydroxide,tetrabutylammonium hydroxide, tetraoctadecylammonium hydroxide,methyltripropylammonium hydroxide, tetrabutylammonium ethoxide,tetraethylammonium hydroxide, tetrahexylammonium hydroxide,tetrakis(decyl)ammonium hydroxide, tetramethylammonium hydroxide,trimethylphenylammonium hydroxide, or a combination thereof.

The metal amide, metal alkoxide, or metal silanoate can be lithiumtert-amoxide, lithium bis(trimethylsilyl)amide, lithium diethylamide,lithium dimethylamide, lithium diisopropylamide, sodiumbis(trimethylsilyl)amide, potassium bis(trimethylsilyl)amide, lithiumdicyclohexylamide, lithium trimethylsilanolate, sodium methoxide,potassium methoxide, lithium methoxide, sodium ethoxide, potassiumethoxide, lithium ethoxide, lithium isopropoxide, sodium tert-butoxide,potassium tert-butoxide, lithium tert-butoxide, sodium tert-pentoxide,potassium tert-pentoxide, magnesium ethoxide, magnesiumdi-tert-butoxide, sodium trimethylsilanolate, potassiumtrimethylsilanolate, or a combination thereof.

The amine can be a cyclic amine of2-(2-chloro-6-fluorophenyl)ethylamine, 1,4-diazabicyclo[2.2.2]octane(DABCO® 33-LV), 1,5-diazabicyclo[4.3.0]non-5-ene,1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]undec-7-ene,4-(dimethylamino)pyridine, 2,6-lutidine, piperidine,1,8-(dimethylamino)naphthalene, 2,2,6,6-tetramethylpiperidine,2,8,9-triisobutyl-2,5,8,9-tetraaza-1-phosphabicyclo[3.3.3]undecane,tripelennamine, aniline, benzylamine, N-methyl aniline, imidazole,pyrrole, pyridine, morpholine, or a combination thereof.

The amine can be a primary amine, a secondary amine, a tertiary amine,or a combination thereof. The primary amine can be methylamine,ethylamine, propylamine, iso-propylamine, butylamine, iso-butylamine,sec-butylamine, tert-butylamine, pentylamine, iso-pentylamine,sec-pentylamine, tert-pentylamine, hexylamine, iso-hexylamine,sec-hexylamine, tert-hexylamine, ethylenediamine, (2-methylbutyl)amine,2-aminopentane, 3-(tert-butoxy)propylamine, 2-amino-6-methylheptane,1-ethylpropylamine, or a combination thereof. Further, the secondaryamine can be dimethylamine, diethylamine, dipropylamine, dibutylamine,dipentylamine, dihexylamine, methylethylamine, methylpropylamine,methylbutylamine, ethylpropylamine, ethylbutylamine, N-ethylmethylamine,N-isopropylmethylamine, N-butylmethylamine, N-ethylisopropylamine,N-tert-butylmethylamine, N-ethylbutylamine, 3-isopropoxypropylamine,chloro(diethylamino)dimethylsilane, 2,2′-(ethylenedioxy)bis(ethylamine),1,3-bis(chloromethyl)-1,1,3,3-tetramethyldisilazane,N-tert-butylisopropylamine, N,N-diethyltrimethylsilylamine,di-sec-butylamine, or a combination thereof. Additionally, the tertiaryamine can be trimethylamine, triethylamine, tripropylamine,tributylamine, dimethylethylamine, dimethylpropylamine,dimethylbutylamine, diethylmethylamine, diethylpropylamine,diethylbutylamine, N,N-diisopropylmethylamine, N-ethyldiisopropylamine,N,N-dimethylethylamine, N,N-diethylbutylamine, 1,2-dimethylpropylamine,N,N-diethylmethylamine, N,N-dimethylisopropylamine,1,3-dimethylbutylamine, 3,3-dimethylbutylamine, N,N-dimethylbutylamine,or a combination thereof.

In various embodiments, the amine is a tertiary amine, for example,trimethylamine, triethylamine, tripropylamine, tributylamine,dimethylethylamine, dimethylpropylamine, dimethylbutylamine,diethylmethylamine, diethylpropylamine, diethylbutylamine,N,N-diisopropylmethylamine, N-ethyldiisopropylamine,N,N-dimethylethylamine, N,N-diethylbutylamine, 1,2-dimethylpropylamine,N,N-diethylmethylamine, N,N-dimethylisopropylamine,1,3-dimethylbutylamine, 3,3-dimethylbutylamine, N,N-dimethylbutylamine,or a combination thereof.

The alkanolamine can be 2-amino-2-(hydroxymethyl)-1,3-propanediol(Trizma® base), propanolamine, ethanolamine, diethanolamine,dimethylethanolamine, N-methylethanolamine, triethanolamine, or acombination thereof.

The conjugate base of a weak acid could be an acetate, a citrate, asuccinate, an oxalate, a malate, a malonate, a phosphate, a phosphonate,a sulfate, a sulfamate wherein the counterion can be a positive ion suchas alkali metal, alkaline earth metal, or ammonium cations.

Unless otherwise indicated, the alkyl groups described herein arepreferably lower alkyl containing from one to eight carbon atoms in theprincipal chain and up to 20 carbon atoms. Alkyls may be substituted orunsubstituted and straight or branched chain. Examples of unsubstitutedalkyls include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl,s-butyl, t-butyl, n-pentyl, i-pentyl, s-pentyl, t-pentyl, and the like.The term “substituted,” as in “substituted alkyl,” means that variousheteroatoms such as oxygen, nitrogen, sulfur, phosphorus, and the likecan be attached to the carbon atoms of the alkyl group either in themain chain or as pendant groups. For example, the substituted alkylgroups can have —C—X—C— fragments in the main chain wherein the X is aheteroatom. Further, the substituted alkyl groups can have at least onehydrogen atom bound to a carbon atom replaced with one or moresubstituent groups such as hydroxy, alkoxy, alkylthio, phosphino, amino,halo, silyl, nitro, esters, ketones, heterocyclics, aryl, and the like.

Having described the invention in detail, it will be apparent thatmodifications and variations are possible without departing from thescope of the invention defined in the appended claims.

EXAMPLES

The following non-limiting examples are provided to further illustratethe present invention.

Example 1 Bench-Scale Packed Bed Reactor

A flow diagram for a packed bed reactor for hydrating CO₂ is shown inFIG. 3. A packed bed reactor having the parts indicated in FIG. 3 wasfabricated. The reactor housing was made of 3 inch diameter PVC pipewith PVC caps on each end. The packing material in the reactor wascommercially available lava rock covered with 0.18 wt. % by weightimmobilized carbonic anhydrase. The immobilized carbonic anhydrase isdescribed below. Carbon dioxide gas entered the reactor through asparger at the base of the reactor and exited to atmospheric pressure atthe top of the reactor. The aqueous solution of 500 mM sodium carbonateentered at the top of the reactor and exited on the side of the reactor.This configuration allowed maintenance of the liquid level in thereactor without use of a second pump.

The carbonic anhydrase was coated onto the packing and then tested.Sample 1A used a 100 mL packing volume and a 239 g packing weight for0.18 wt. % carbonic anhydrase. A solution of 428 mg bovine carbonicanhydrase, 0.425 mL Triton X-100, 84.575 mL 0.02M Trissulfate buffer atpH 8.3, and 15 mL 15% tetraethylammonium bromide-modified Nafion® wasprepared to form an immobilized enzyme solution. The solution wasvortexed for approximately five minutes. The enzyme immobilizationsolution was pipetted onto the lava rock and allowed to dry overnight at4° C. and then placed in a vacuum oven at room temperature and −30 mmHgfor 2 hours before placing the lava rock into the reactor. Sample 1B hadthe same packing volume and packing weight, but contained no carbonicanhydrase for use as a control.

The reactor inlet flow rates, compositions of gas and liquid streams,packing weight, catalyst weight, and mol CO₂/(h L) sequestered werecollected. The mol CO₂/(h L) sequestered was calculated based on theamount of sodium hydroxide/potassium hydroxide used to reclaim thecarbonate solution through the pH stat. The overall system isillustrated in FIG. 3. A 0.5 M sodium carbonate (Na₂CO₃) solution at a4.18 L/h and a CO₂ flow rate of 7308 L/h was used in the reaction (Thereactor conditions were not optimized).

When using the pH stat to determine how much carbon dioxide wassequestered, the dosing unit added a volume of 2 M sodium hydroxide(NaOH) in a recorded amount of time. A graph of the volume (mL) ofsodium hydroxide added per unit time(s) with and without immobilizedcarbonic anhydrase are given in FIGS. 4 and 5, respectively.

The experiment wherein the packing material was coated with immobilizedcarbonic anhydrase showed that 0.719 mol/h of carbon dioxide wassequestered whereas the experiment wherein the packing material was notcoated with enzyme showed that 0.471 mol/h of carbon dioxide wassequestered. The amount of carbon dioxide removed is calculated by theequation

${{{mol}\; {CO}_{2}} = {{\left( \frac{\Delta \; V}{\Delta \; t} \right)\left\lbrack {{Na}\; {OH}} \right\rbrack}\left( \frac{{mol}\; {CO}_{2}}{2\mspace{14mu} {mol}\; {Na}\; {OH}} \right)}},$

where ΔV is the change in volume and Δt is the change in time.

Example 2 Dehydration of Bicarbonate Solutions Using Carbonic Anhydrase

The rate of reaction for conversion of bicarbonate ions to CO₂, waterand carbonate ions was monitored by the change in pH as a function oftime in a closed system. The system parameters that were altered includetemperature of the reaction, initial bicarbonate concentration, andreaction time. The experiments were carried out in a 200 mL three neckedflask containing 80 mL of sodium bicarbonate solution with 0.313 mg/mLcarbonic anhydrase either free in solution or immobilized on a poroussupport added to the solution. The temperature of the system was set toa constant set point using a thermostatically controlled water bath.Nitrogen gas was introduced into the system at a rate of 50 standardcubic centimeters per minute (sccm) through a fritted glass sparger ofmedium porosity to remove any carbon dioxide produced during the courseof the reaction. The initial pH of the system was approximately 8.4 andthe change in pH was monitored by a temperature compensated pH probe andthe data was logged at three second intervals. All enzymatic systemstested were compared to comparable blanks of either a bicarbonatesolution containing no enzyme or a bicarbonate solution containingsupport material having no immobilized enzyme coated thereon.

Example 2A Free Enzyme in a Bicarbonate Solution

Dehydration of bicarbonate catalyzed by non-immobilized carbonicanhydrase was compared to dehydration of bicarbonate without a catalyst.A graph of the pH versus the reaction time comparing the carbonicanhydrase catalyzed reaction to the uncatalyzed system is shown in FIG.6. The temperature of this reaction was set at 40° C. and the initialconcentration of the bicarbonate in solution was 0.1 M. The solution wasstirred using an overhead glass rod fitted with apolytetrafluoroethylene (PTFE) paddle. Data collection for the reactionwas initiated when the enzyme was introduced into the system and wascontinuously collected for approximately 50 minutes. Initially, theenzymatic system demonstrated a higher rate of pH change for the overallsystem when compared to the blank and the faster rate continuedthroughout the entire experiment. At 45 minutes, the pH of theuncatalyzed reaction mixture was 8.9 compared to over 9.2 for thecarbonic anhydrase catalyzed reaction mixture after the same elapsedtime. The carbonic anhydrase-catalyzed reaction mixture had a 0.5 pHunit increase in one third of the time as compared to the uncatalyzedreaction.

When the reaction was allowed more time for completion (up to 16 hours),the carbonic anhydrase-catalyzed reaction mixture maintained a higher pHvalue than the uncatalyzed reaction mixture throughout the entireexperiment. A graph of the pH of the reaction mixture versus time forthe 16 hour experiment is shown in FIG. 7. The carbonic anhydrasesolution ended at a pH of 10.1 after 16 hours. A sodium carbonatesolution has a pH of approximately 11.3, so the concentration ofcarbonate was higher in the reaction mixture that was allowed to reactlonger.

Example 2B Varying the Bicarbonate Concentration

The initial concentration of bicarbonate was varied between values of0.1 M and 2.0 M for the non-immobilized enzyme system. FIG. 8 is a graphof the pH versus the reaction time for three carbonic anhydrasecatalyzed reaction mixtures having an initial bicarbonate concentrationof 0.1, 0.5, and 2.0 M. The largest change in pH was observed in thelowest concentration system. As the concentration of bicarbonateincreased to 0.5 M the change in the pH of the system was comparable tothat of the blank system at 0.1 M, but still slightly better than theblank at 0.5 M. The rate of pH increase and the final pH value slowedonly slightly when the concentration of the bicarbonate was increased to2.0 M when compared to the 0.5 M system. The inverse relation of thesystem response to increasing bicarbonate levels could have many causes.For example, the buffering capacity of the bicarbonate/carbonatesolution increases as the concentration of the system increases becausebicarbonate is converted to carbonate, thus causing the pH not to riseas fast as it would without such buffering. Further, the particularstrain of bovine carbonic anhydrase used may favor the carbon dioxidehydration reaction over that of the bicarbonate dehydration reaction andas the reaction progressed, the carbonic anhydrase preferentiallyconverted the newly formed carbonate and carbon dioxide back to thebicarbonate form. Finally, the combined effects of higher ionicconcentrations and elevated temperatures has been shown to reduce theactivity of free carbonic anhydrase in the carbon dioxide hydrationreaction and a similar result might be expected for the bicarbonatedehydration.

Example 2C Reaction Temperature

For an uncatalyzed reaction the rate of bicarbonate dehydrationincreases with increasing temperature of the reaction mixture. This wasaccomplished for the conversion of bicarbonate to carbonate by passingsteam through the reaction mixture to increase the reaction temperatureand thus, reaction rate, and to remove the carbon dioxide produced fromthe solution. A graph of the pH versus time for a carbonic anhydrasecatalyzed conversion of bicarbonate to carbonate, CO₂, and water at 20°C. and 40° C. in a 0.1 M bicarbonate solution is shown in FIG. 9. Forthe non-immobilized enzyme system, the rate of reaction was slightlyfaster for the room temperature system over that of the highertemperature system. This may be the result of the combined effects ofthe high temperature and ionic concentration reducing the activity ofthe enzyme, similar to the results of the bicarbonate concentrationstudies previously.

Example 2D Immobilized Carbonic Anhydrase

A graph of the pH versus time for the dehydration of bicarbonate usingan immobilized carbonic anhydrase as a catalyst as compared to anuncatalyzed reaction mixture are shown in FIG. 10. The carbonicanhydrase in this example was encapsulated in tetraethylammonium bromidemodified Nafion® onto washed and crushed lava rock supports (<1 cm) at aloading of 0.5% wt with 0.5% wt Triton X surfactant to maintain theenzyme hydration throughout the drying process.

The immobilized carbonic anhydrase was prepared as follows. Ethanol (2mL) was added to tetraethyl ammonium bromide (TEAB) modified Nafion® (30mg) to make a 5.0 wt. % solution. Carbonic anhydrase (50 mg) was addedto 2 mL Trizma Base buffer solution (0.05 M, pH 7.6) to which 0.02 mLTriton X-100 was added at a total solution percentage of 0.5% andstirred until a uniform dissolution occurred. Once the solution wasadequately dispersed, the TEAB-modified Nafion® solution was added andstirred until the solution was sufficiently homogenous. Once theimmobilized enzyme solution was thoroughly mixed it was cast onto highsurface area support and allowed to dry for 12 hours at 4° C. followedby 2 hours under vacuum. Alternatively, high surface carbon support wasadded to the immobilized enzyme solution, mixed, sprayed, and allowed todry for several hours at room temperature.

The activity and performance of the immobilized enzyme was very similarto that of the free enzyme at the same salt concentration andtemperature. Future testing on this sample will include exposure tohigher temperatures (>50 C), higher concentrations of bicarbonate, andoperating lifetime compared to non-immobilized enzyme. The same packingmaterial with immobilized carbonic anhydrase was used for Run 1 and Run2. This difference in performance may be attributed to the stability ofthe immobilized enzyme leaching after multiple washes and exposures tohigh carbonate concentration solutions.

Example 3 Immobilization of Carbonic Anhydrase TBAB-Modified Nafion® onPrintex-95 Carbon

Bovine carbonic anhydrase (70 mg, purchased from Sigma-Aldrich) wascombined with 10 mL of 20 mM Tris-SO₄ buffer of pH 8.3 and vigorouslyvortexed for approximately 5 seconds. Printex-95 carbon support material(0.5 gram) was combined with the enzyme solution and vigorously vortexedfor one minute at room temperature. Tetrabutylammonium bromide(TBAB)-modified Nafion® (2 mL; 15% w/v) in 95% ethanol solution wasadded to the enzyme/support suspension and vigorously vortexed for oneminute at room temperature. The enzyme/support/Nafion®/modifier solutionwas spray dried onto a mirror with nitrogen gas at about 20 psi. Theresulting immobilized sample was allowed to dry at room temperature onthe mirror for 30 minutes. Dried immobilized enzyme was scraped off themirror and stored at about 4° C.

Enzyme activity was measured and calculated using a carbonic anhydraseassay as published by Sigma (revision date Jul. 22, 1996). The assaymeasures the rate of enzymatic CO₂ hydration by determining the net ratedifference between a non-enzymatic blank and an enzyme-containing samplein the time required to decrease the pH of a buffered reaction mixturefrom 8.3 to 6.3. This enzyme activity assay was used in this and allsubsequent examples.

Data obtained showed no net increase in reaction rate using immobilizedenzyme relative to the non-enzymatic reaction. The amount of enzyme usedin preparation and testing of the immobilized material, if active, wasexpected to show a pronounced rate increase.

Example 4 Immobilization of Carbonic Anhydrase in TetraethylammoniumBromide (TEAB)-Modified Nafion® on Printex-95 Carbon

Carbonic anhydrase was immobilized as described for Example 3 exceptthat tetraethyl ammonium bromide was used as the modifier. Again, no netincrease in rate was observed in the assay of this immobilized enzyme.The amount of enzyme used in preparation and testing of the immobilizedmaterial, if active, was expected to show a pronounced rate increase.

Example 5 Immobilization of Carbonic Anhydrase in Hexanal-ModifiedChitosan on Poly(styrene-co-divinylbenzene) (PS-coDVB)

Carbonic anhydrase was immobilized as described in previous examplesexcept that the polymer and modifier were chitosan and hexanal,respectively and the enzyme/polymer/modifier suspension was mixed withbefore spray drying. There was no net increase in the rate of CO₂hydration over the non-enzymatic control reaction. The amount of enzymeused in preparation and testing of the immobilized material, if active,was expected to show a pronounced rate increase.

Example 6 Immobilization of Carbonic Anhydrase in DeacetylatedChitosan/Acetaldehyde on PS-co-DVB

Carbonic anhydrase was immobilized and spray dried as described inprevious examples except that the polymer and modifier were deacetylatedchitosan and acetaldehyde. The support waspoly(styrene-co-divinylbenzene) of 8 μm nominal particle size. No netenzyme activity was observed in this immobilization material. The amountof enzyme used in preparation and testing of the immobilized material,if active, was expected to show a pronounced rate increase.

Example 7 Immobilization of Triton X-100 Treated Carbonic Anhydrase inTEAB-Modified Nafion® on Printex-95 Carbon

Bovine carbonic anhydrase (70 mg; purchased from Sigma-Aldrich) wascombined with 10 mL of 20 mM Tris-SO₄ of pH 8.3 buffer and 0.050 mLTriton X-100. The immobilization and spray drying were done as inExample 2. Triton-treated enzyme immobilized by this procedure wastested for activity and had 1077 units of activity per gram materialcontaining immobilized enzyme.

Example 8 Immobilization of Decylamine-Modified Carbonic Anhydrase inTEAB-Modified Nafion® on Printex-95 Carbon

Carbonic anhydrase (70 mg) was combined with 5 mL of 75 mM MES buffer ofpH 5.0 and then 7.8 mg N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (EDC) (4 mM) and 23.8 mg N-hydroxysulfosuccinimide sodiumsalt (sulfo-NHS) (11 mM). The solution was vigorously vortexed for fiveseconds. A second solution was made with 5 mL of 75 mM MES buffer of pH5.0 was combined with 7.87 mg decylamine. This solution was combinedwith the EDC/enzyme solution and vigorously vortexed for 5 seconds. Thecombined solutions were held refrigerated overnight. Then the modifiedenzyme was mixed with 15% (w/v) TEAB-modified Nafion® and 1 gramPrintex-95 and spray dried as described above. The enzyme activity assayshowed that this modification had 3729 units of activity per grammaterial containing immobilized enzyme.

Example 9 Immobilization of (PEG) 8-Modified Carbonic Anhydrase onPrintex-95 Carbon

Carbonic anhydrase (70 mg) was dissolved and used as described inExample 8 except that instead of decylamine, 11.0 mg (PEG) 8-modifiedcarbonic anhydrase from Pierce Scientific was used. After overnightrefrigeration, the modified enzyme preparation was combined withPrintex-95 carbon and then spray dried as in Example 8. The enzyme assayshowed that this immobilized modified carbonic anhydrase preparation had3421 units of activity per gram material containing immobilized enzyme.

Example 10 Carbonic Anhydrase Immobilized in Polysulfone

When immobilizing carbonic anhydrase in polysulfone, carbonic anhydrasewas dissolved in 8 g 1-methylpyrrolidone and 0.05 mL Triton X-100. Thepolysulfone (2 g; 20 wt. %) was added to the solution and stirred untilcompletely dissolved. The polysulfone/carbonic anhydrase solution washeld at room temperature until complete mixing was achieved. Thedissolved polysulfone/carbonic anhydrase solution was then addeddropwise to a water, an alcohol, or a water-alcohol solution and formedpolymeric beads (Sample 1A) as shown in FIG. 11. Blue beads (Sample 1B)were prepared by mixing 200 mg of copper phthalocyanine into 10 mL ofthe 20 wt. % polysulfone in 1-methylpyrrolidone solution. Next, thesolution was added dropwise into a beaker of water, thus forming thebeads. The beads were washed repeatedly with water, alcohol, andcarbonate solution to wash any free dye off the bead.

Sample 1C was prepared by dissolving 100 mg carbonic anhydrase in 10 mL1-methylpyrrolidone with 0.05 mL Triton X-100 and adding 20 wt. %polysulfone in 1-methylpyrrolidone. Samples 1A and 1B are pictured inFIG. 11. Sample 1C was tested for enzymatic activity. FIG. 12 shows thecomparison of carbonic anhydrase immobilized in polysulfone to blanksolution and free enzyme in solution. The immobilized enzyme showedactivity, but the activity was not a high as that of free enzyme. Theexperiment used beads of Sample 1C placed in 0.5 M sodium carbonate at0° C. in a three ring flask with 50 sccm carbon dioxide sparged into thesolution. The pH was monitored using a temperature adjusted pH meterover the course of approximately 50 minutes. Each run was started whenthe pH reached 11.0 in order to have an adequate pH verse timecomparison.

Example 11 Carbonic Anhydrase Immobilized Polysulfone/AlginateCore-Shell Particulate Support

Carbonic anhydrase was immobilized in alginate to form alginate beads bymixing 25 mg carbonic anhydrase with 2 mL of a 2 wt. % alginatesolution. This solution was added dropwise into 50 mL of a 2 wt. %calcium chloride solution and stirred for at least 30 minutes. The beadshaving carbonic anhydrase immobilized in alginate were then removed andgently dried with paper towels. Next, the beads were rolled into a 20wt. % polysulfone solution by hand to obtain a thin polysulfone filmcoating the alginate beads. Alginate beads dissolve in sodium carbonatesolution without the polysulfone coating. But, the alginate does notdissolve and the carbonic anhydrase is retained when a thin film ofpolysulfone coats the bead. This process could also be used for otherpolymeric immobilization materials to advantageously control thesubstrate diffusion.

FIG. 13 shows the comparison of carbonic anhydrase immobilized inalginate encapsulated in polysulfone to blank solution. The immobilizedenzyme did show activity throughout both runs. The first run was donealmost immediately after the alginate beads were coated withpolysulfone. The second run was soaked in a sodium carbonate/sodiumbicarbonate solution overnight and then washed and tested in freshsolution. The resulting data suggested a diffusional limitation on theoverall carbon dioxide reacted. As was seen in FIG. 13, the first runhas a lower reaction rate than the blank for the first 20 minutes of thereaction, while the second run closely corresponds to the blank, butboth runs containing enzyme end the experiment at the same pH value. Theexperiment consisted of 0.5 M sodium carbonate at 0° C. in a three ringflask with 50 sccm carbon dioxide sparged into the solution. The pH wasmonitored using a temperature adjusted pH meter over the course ofapproximately 50 minutes. Each run was started when the pH reached 11.0in order to have an adequate pH verse time comparison.

Example 12 Carbonic Anhydrase Immobilized in Modified Polyvinyl BenzylChloride)

FIG. 14 shows the activity of this diamine cross-linked PVBCencapsulated carbonic anhydrase is approximately 50% that of the freeenzyme in solution. The sample shown in FIG. 14 was taken after 2 hoursof vigorous stirring in distilled, deionized water, demonstratedretention of enzyme within the support.

First, 5 mL of a 20 wt. % solution of poly(vinylbenzyl chloride) indioxane was prepared. Then, 80 mg carbonic anhydrase was added to thesolution and stirred until there was uniform dissolution. Once thesolution was adequately dispersed, 0.435 mL N, N,N′,N′-tetramethyldiaminomethane was added and stirred until the solution was sufficientlyviscous to form a bead. However, the stirring was not long enough toallow the solution to gel. When the viscosity was low enough to beeasily pipetted using a transfer pipette, the solution was addeddropwise into a beaker of water and stirred to remove the excesssolvent.

Example 13 Synthesis of Aminated Polysulfone

Polysulfone (10 g, PSf) and 40 mL of 1,2-dichloroethane were placed in a3-neck 250 mL round bottom flask, and the solution was stirred with aTeflon stir bar to dissolve polysulfone. Once homogenized, 20 mLchloromethyl methyl ether and 2 g zinc chloride (ZnCl₂) were added tothe flask. The flask was equipped with a thermometer, a condenser, and arubber septum to cover the third opening. The reaction mixture was thenheated to 40° C. with stirring and reacted for 4.5 hours. The solutionwas then cooled to room temperature and precipitated into 1.2 L ofmethanol. The crude chloromethylated polysulfone was collected and driedin the vacuum oven overnight at room temperature. This polymer was thenredissolved in 200 mL 1,4-dioxane and reprecipitated into 1.2 L ofmethanol. The purified chloromethylated polysulfone (PSf-CH₂Cl) was thencollected and dried in the vacuum oven overnight at room temperature. ¹HNMR results indicated that 33% of the benzene rings in the polysulfonebackbone were chloromethylated, corresponding to an average of 1.3chloromethyl groups per repeat unit. A 20 wt. % solution of PSf-CH₂Cl in1,4-dioxane was prepared by stirring with a Teflon stir bar in a glassvial.

Amination via trimethylamine. Beads of PSf-CH₂Cl were prepared byprecipitating the 20 wt. % PSf-CH₂Cl in dioxane solution into a beakerwith 500 mL deionized water. The beads were then stirred in thedeionized water with a Teflon stir bar for 30 minutes. The beads werethen collected and soaked in a solution of 0.04M trimethylamine indeionized water for 24 hours. They were then collected, rinsed withdeionized water, and soaked in a 1M potassium hydroxide or potassiumbicarbonate aqueous solution for 24 hours to exchange the chlorideanions for either hydroxide or bicarbonate ions.

Amination via a tertiary diamine. A tertiary diamine (such asN,N,N′,N′-tetramethyl-1,6-hexanediamine, TMHDA) was added to the 20 wt.% PSf-CH₂Cl in dioxane solution at an equimolar ratio of chloromethylgroups to tertiary nitrogens (equivalent to a 1:0.5 ratio ofchloromethyl groups to diamine). For instance, 0.32 mL of TMHDA wasadded to 5 mL of a 20 wt. % solution of PSf-CH₂Cl described above (1.3chloromethyl groups per repeat unit). The mixture was stirred forseveral minutes until noticeably more viscous. Beads were then preparedby precipitating this mixture into a beaker with 500 mL deionized water.The beads were then stirred in the deionized water with a Teflon stirbar for 30 minutes. They were then collected and soaked in a 1Mpotassium hydroxide or potassium bicarbonate aqueous solution for 24hours to exchange the chloride anions for either hydroxide orbicarbonate ions.

Example 14 Synthesis of Aminated Polycarbonate

Polycarbonate (10 g, PC) and 80 mL of 1,2-dichloroethane were placed ina 3-neck 250 mL round bottom flask, and the solution was stirred with aTeflon stir bar to dissolve polycarbonate. Once homogenized, 20 mLchloromethyl methyl ether and 2 g zinc chloride (ZnCl₂) were added tothe flask. The flask was equipped with a thermometer, a condenser, and arubber septum to cover the third opening. The reaction mixture was thenheated to 40° C. while stirring and reacted for 4.5 hours. The solutionwas then cooled to room temperature and precipitated into 1.2 L ofmethanol. The crude chloromethylated polycarbonate was collected anddried in the vacuum oven overnight at room temperature. This polymer wasthen redissolved in 200 mL 1,4-dioxane and reprecipitated into 1.2 L ofmethanol. The purified chloromethylated polycarbonate (PC-CH₂Cl) wasthen collected and dried in the vacuum oven overnight at roomtemperature. ¹H NMR results indicated that only 5% of the benzene ringsin the polycarbonate backbone were chloromethylated, corresponding to anaverage of 0.1 chloromethyl groups per repeat unit. A 20 wt. % solutionof PC-CH₂Cl in N-methylpyrrolidone (NMP) was prepared by stirring with aTeflon stir bar in a glass vial.

Amination via trimethylamine. Beads of PC-CH₂Cl were prepared byprecipitating the 20 wt. % PC-CH₂Cl in NMP solution into a beaker with500 mL deionized water. The beads were then stirred in the deionizedwater with a Teflon stir bar for 30 minutes. The beads were thencollected and soaked in a solution of 0.04M trimethylamine in deionizedwater for 24 hours. They were then collected, rinsed with deionizedwater, and soaked in a 1M potassium hydroxide or potassium bicarbonateaqueous solution for 24 hours to exchange the chloride anions for eitherhydroxide or bicarbonate ions.

Amination via a tertiary diamine. A tertiary diamine (such asN,N,N′,N′-tetramethyl-1,6-hexanediamine, TMHDA) was added to the 20 wt %PC-CH₂Cl in NMP solution at an equimolar ratio of chloromethyl groups totertiary nitrogens (equivalent to a 1:0.5 ratio of chloromethyl groupsto diamine) For instance, 0.04 mL of TMHDA was added to 5 mL of a 20 wt.% solution of PC-CH₂Cl described above (0.1 chloromethyl groups perrepeat unit). The mixture was stirred for several minutes untilnoticeably more viscous. Beads were then prepared by precipitating thismixture into a beaker with 500 mL deionized water. The beads were thenstirred in the deionized water with a Teflon stir bar for 30 minutes.They were then collected and soaked in a 1M potassium hydroxide orpotassium bicarbonate aqueous solution for 24 hours to exchange thechloride anions for either hydroxide or bicarbonate ions.

Example 15 Synthesis of Crosslinked Poly(vinylbenzyl chloride)

A 33 wt. % solution of poly(vinylbenzyl chloride) (PVBC) in dioxane wasprepared by stirring with a Teflon stir bar in a glass vial. The choiceof tertiary diamine or tertiary diamine mixture utilized tosimultaneously aminate and crosslink PVBC affects the resulting chemicaland mechanical properties of the beads and must be optimized for bestperformance. The use of two different diamine crosslinkers is describedbelow.

Crosslinking with N,N,N′,N′-tetramethyl-methanediamine (TMMDA). TMMDA(0.74 mL) was added to 5 mL of 33 wt. % PVBC dioxane solution(corresponding to an equimolar ratio of chloromethyl groups tonitrogens). The mixture was stirred for 3 minutes until noticeably moreviscous. Beads were then prepared by precipitating this solution into abeaker with 500 mL deionized water. The beads were then stirred in thedeionized water with a Teflon stir bar for 30 minutes. They were thencollected and soaked in a 1M potassium hydroxide or potassiumbicarbonate aqueous solution for 24 hours to exchange the chlorideanions for either hydroxide or bicarbonate ions.

Crosslinking with N,N,N′,N′-tetramethyl-phenylenediamine (TMPDA). TMPDA(0.89 g) was added to 5 mL of 33 wt. % PVBC dioxane solution(corresponding to an equimolar ratio of chloromethyl groups tonitrogens). The mixture was stirred for 1 hour until noticeably moreviscous. The reaction of PVBC with TMPDA was much slower than itsreaction with TMMDA, so these solutions were stirred longer before beadformation. Beads were then prepared by precipitating this solution intoa beaker with 500 mL deionized water. The beads were then soaked in thedeionized water for 30 minutes. These beads were not stirred in waterafter precipitation because these beads were hydrophilic, high-swellingmaterials that could break apart with strong agitation. They were thencollected and soaked in a 1M potassium hydroxide or potassiumbicarbonate aqueous solution for 24 hours to exchange the chlorideanions for either hydroxide or bicarbonate ions.

Example 16 Carbonic Anhydrase Immobilized in Aminated Polysulfone

FIG. 5 shows the best retention of activity seen for aminated polymericimmobilization materials. The activity of this aminated polysulfoneimmobilized carbonic anhydrase is approximately 70% that of the freeenzyme in solution. The sample shown in FIG. 5 was taken after 2 hoursof vigorous stirring in DI water and demonstrated retention of enzymewithin the immobilization material. To make the immobilized carbonicanhydrase particles, a solution of 5 ml of 20 wt. % chloromethylatedpolysulfone was made in dioxane. Then 50 mg carbonic anhydrase was addedto the solution and stirred until a uniform dissolution occurred. Oncethe solution was adequately dispersed, tetramethyl diamine was added andstirred until the solution was sufficiently viscous to form a bead.However, the stirring was not long enough to allow the solution to gel.When the viscosity was low enough to be easily pipetted using a transferpipette, the solution was added dropwise into a beaker of water andstirred to remove excess solvent.

Example 17 Hydration of CO₂ in Amine Solutions Using Carbonic Anhydrase

An aqueous solution containing water, an amine, and in some cases anenzyme had carbon dioxide bubbled through it and the amount of carbondioxide that was captured was measured by determining the amount ofsodium hydroxide added to the solution in order to maintain a pH valueof 11 (the pH of the amine solutions). However, one MDEA test wasperformed at pH 8.4 (pH adjusted by sparging solution with carbondioxide). Carbon dioxide was introduced into the aqueous solution at arate of 200 sccm through a spherical sparging stone.

The immobilization material used for the was a polysulfone (PSf)backbone with polyethyleneoxide (PEO; average molecular weight of 550)grafted to a degree of functionality of 0.5 which corresponds toapproximately 38% wt. (PEGylated PSf). The carbonic anhydrase (bovineCA) was immobilized in PEGylated PSf spheres using the followingprocess. The enzyme was dissolved into the solvent with a surfactant. Toencapsulate carbonic anhydrase in a polysulfone, the solvent wasselected to not deactivate the enzyme. The polymer was added to thesolution and stirred until completely dissolved. The polymer enzymesolution was held at room temperature until completely mixed. Thedissolved polymer enzyme solution was then added dropwise to water,alcohol, or a water-alcohol solution creating polymeric beads. Theenzyme used was a commercially available mammalian CA from a bovinesource.

The primary amine used for these studies was monoethanolamine (MEA) thatwas tested at concentrations of 12 and 144 mM in DI water with an enzymesolution loading for the enzyme free in solution of 0.25 mg/mL in bothamine concentrations and 0.5 mg/mL in the 144 mM MEA solution and animmobilized enzyme loading of 0.25 mg/mL solution. The tertiary alcoholamine that was examined is N-methyldiethanolamine (MDEA) and was testedat concentrations of 12 and 144 mM in DI water with an enzyme solutionloading of 0.25 mg/mL in both amine concentrations. A second non-alcoholtertiary amine was included in this study, N,N-diethylmethylamine (DMA),and was tested at concentrations of 71 and 144 mM in DI water with anenzyme solution loading of 0.25 mg/mL in both amine concentrations. Allof the solutions were kept at room temperature and the total volumeswere set at 50 mL.

The enzyme contribution was separated from the amine contributionthrough the comparisons of amine solutions to amine plus enzymesolutions. The results of this study, in terms of total carbon dioxidecaptured for both the amine and enzyme component, are shown in FIG. 16.The total amount of carbon dioxide captured with both enzyme and aminewas relatively the same for all systems at pH 11. The enzyme had thegreatest contribution of approximately 25% in the 12 mM MDEA and 12 mMMDEA/12 mM MEA solutions. The enzymes had the least contribution tototal carbon dioxide capture in the DMA solutions, but the overallcarbon dioxide capture in these solutions was the largest of all thesystems tested and at the concentrations tested, seemed to beindependent of amine concentration.

The specific activities of enzyme in all of the above listed conditionsare shown in FIG. 17. The largest activity of the enzyme system wasobserved in the mixed MEA and MDEA solution followed by the lowconcentration MDEA solution. The enzyme had the highest specificactivity in the low concentration alcohol amine solutions when comparedto the higher concentrations of the same amine. It is unclear whetherthese enzyme activities are the result of substrate inhibition by theamine or because the amine base is competitive in absorbing the carbondioxide at higher concentrations.

Example 18 Removal of Carbon Dioxide from Solution Using CarbonicAnhydrase

Carbonic anhydrase was used for removing carbon dioxide from the aqueousliquid described in example 17. The experimental design and conditionsof this study were similar to Example 17, but the substrate for thecarbonic anhydrase was the absorbed carbon dioxide and the immobilizedenzyme mixture was cast onto a high surface area support, crushed lavarock (about 1 cm in diameter), and allowed to dry into a film.

The reaction of carbon dioxide in an amine solution can producecarbamates, carbonate, bicarbonate, or mixtures depending on thereaction temperature, the nature of the amine (i.e., primary, secondary,or tertiary amine), the carbon dioxide partial pressure, and thereaction pH. It was determined that generating the substrate byintroducing carbon dioxide gas into the system for a fixed amount oftime to produce the substrate and then monitoring the change in solutionpH to follow the release of carbon dioxide was an efficient way toconduct the experiment. The concentration of the amine, MEA and MDEA,was 0.05 M, the reaction was tested at room temperature and at 50° C.,and the enzyme mass was 25 mg. The products for the reverse reactionwere generated by introducing carbon dioxide through a glass fritsparger into the amine solution, with and without enzyme, at a rate of200 sccm for 10 minutes. The pH of this reaction was monitored to verifythat the conversion of the amine was complete and that the starting pHof the reverse reaction and the temperature was the same for each sample(e.g., blank amine solution, amine solution with non-immobilized enzyme,and amine solution with immobilized enzyme). After the carbon dioxideexposure, nitrogen gas was introduced into the solution via a glass fritsparger at a rate of 200 sccm for 20 minutes while the solution pH wasmonitored and recorded. The relative activity of the enzyme in each casewas determined by the rate of the pH increase from the starting point tothe final pH of the solution after 20 minutes. The faster the pHincreased and the greater the final pH, the higher the relative activityof the enzyme. The results of the MEA solution at 20° C. and 50° C. areshown in FIGS. 18 and 19, respectively. At 20° C., both the free enzymein solution and the immobilized enzyme had higher conversions of carbondioxide release than the comparable solution with no enzyme. Theimmobilized enzyme had a lower activity than the free enzyme probablydue to mass transfer effects from the immobilization material. At 50°C., there was no difference between the free enzyme and the blanksolution and a reduced performance of the immobilized enzyme wasobserved.

The results of the MDEA solution at 20° C. and 50° C. are respectivelyshown in FIGS. 20 and 21. At 20° C., both the free enzyme in solutionand the immobilized enzyme had higher conversions of carbon dioxiderelease than the comparable solution with no enzyme. The rate of changein solution pH was less for the immobilized enzyme compared to the freeenzyme in solution at the beginning of the experiment but near the endof the experiment the pH of both systems was nearly the same in the MDEAsolution. There was no difference between the free enzyme in the MDEAsolution and the blank MDEA solution and a lower amount of carbondioxide was released in the sample with the immobilized enzyme.

Under the reaction conditions, the reaction of a primary amine withcarbon dioxide produces a carbamate; it is thought that carbonicanhydrase cannot use carbamate as a substrate. Thus, the improvedperformance of the enzymatic system at lower temperatures and not athigher temperatures for this system may be attributed to the initialenzymatic hydration and dehydration of the carbon dioxide in thealkaline solution for both the forward and reverse reactions. Thisreaction appears to dominate the amine reaction at lower temperaturesand the amine reaction becomes dominant as the temperature is increased.

Example 19 Thermal Stability of Carbonic Anhydrase

The thermal stability of several carbonic anhydrases was studied bymeasuring the carbon dioxide activity. The thermal stabilities of bovinecarbonic anhydrase II (BCA II, (purified and unpurified) and humancarbonic anhydrase IV (HCA IV) were determined by incubating 0.2 mg/mLsolutions in deionized (DI) water (25 mL total) for the allotted time inthe oven at 70° C. and then diluting them with equal volume of 0.4MNaHCO₃ to perform the pH stat analysis of carbon dioxide activity. Itwas determined that it takes 1.5 hours for 25 mL of DI water to reach70° C. in the oven, so the total time the samples were in the oven wasadjusted to account for the long lag time in reaching the temperatureset point. These results are summarized in FIG. 22. As seen in thisfigure, HCA IV exhibited a higher thermal stability by retaining nearly100% of its initial activity after 32 hours of exposure at 70° C. Incontrast, BCA II (unpurified) lost all of its activity after only 1 hourat 70° C. Purification of BCA II did improve its thermal stability, butpurified BCA II still had lower thermal stability than HCA IV.

Additional measurements of the thermal stability of HCA IV were limitedby the aggregation of the enzyme seen after 32 hours of exposure to 70°C. The HCA IV appeared to irreversibly aggregate into large chunks ofenzyme that were still active by the carbon dioxide activity test viathe pH stat.

Example 20 Synthesis of Polysulfone Grafted with Polyethylene Glycol(PSf-g-PEG) Immobilization Material

Chloromethylation of Polysulfone. Polysulfone (PSf; 20 g) and 200 mL of1,2-dichloroethane were placed in a 2-neck 500 mL round bottom flask andthe solution was stirred with a Teflon stir bar to dissolve PSf. Oncehomogenized, 15 mL of chloromethyl methyl ether and 1.5 g of zinc(II)chloride (ZnCl₂) were added to the flask. The flask was equipped with athermometer and a condenser. The reaction mixture was then heated to 40°C. while stirring and reacted for a set period of time. The reactiontime determined the chloromethylation degree of PSf. For instance, areaction time of 2.5 hours resulted in a degree of functionalization(DF) of 0.55, meaning an average of 0.55 chloromethyl groups per PSfrepeat unit. Similarly, reaction times of 2.25 hours, 2 hours, and 1.25hours resulted in DFs of 0.5, 0.4, and 0.23, respectively. After theallotted reaction time, the reaction mixture was cooled to roomtemperature, diluted with 200 mL of 1,2-dichloroethane, and precipitatedinto 4.5 L of methanol. The chloromethylated polysulfone (PSf-CH₂Cl) wascollected via filtration and dried in the vacuum oven overnight at roomtemperature.

Poly(ethylene glycol) Grafting onto Chloromethylated Polysulfone. Oncedry, chloromethylated polysulfone (PSf-CH₂Cl; 10 g) and 250 mL of dry1,4-dioxane (dried over molecular sieves) were added to a 500 mL roundbottom flask, and the solution was stirred with a Teflon stir bar todissolve PSf-CH₂Cl. Once homogenized, this flask was capped with arubber septum and flushed with nitrogen for 15 minutes. To a separate 50mL round bottom flask, a 1.5 molar excess (with respect to thechloromethyl groups of PSf-CH₂Cl) of poly(ethylene glycol) monomethylether (PEG) with a molecular weight of 550 Da was added. Sufficient dry1,4-dioxane was added to this flask to make a 25 vol. % PEG solution.For example, for PSf-CH₂Cl with a DF of 0.55, 10.3 mL of PEG and 31 mLof 1,4-dioxane were added. This flask was then capped with a rubberseptum and flushed with nitrogen for 10 minutes. To a separate 100 mLround bottom flask, a 1.5 molar excess (with respect to the chloromethylgroups of PSf-CH₂Cl) of sodium hydride (NaH) was added. Sufficient dry1,4-dioxane was added to this flask to make a 1 wt./vol. % NaH solution.For example, for PSf-CH₂Cl with a DF of 0.55, 0.44 g of NaH and 44 mL of1,4-dioxane were added. This flask was then equipped with a Teflon stirbar, capped with a rubber septum, and flushed with nitrogen for 10minutes. The PEG solution was then added dropwise via a cannula to theNaH solution while stirring. This reaction mixture was then stirred for3 hours at room temperature while venting periodically with a needle torelease generated hydrogen gas. After 3 hours, the PEG/NaH solution wasadded dropwise via a cannula with stirring to the PSf-CH₂Cl solution.This reaction mixture was then stirred for 2 days at room temperature.After the reaction was complete, the reaction mixture was neutralized topH 7 using concentrated acetic acid and then precipitated into 4.5 L ofdeionized water. Polysulfone grafted with PEG (PSf-g-PEG) was thencollected via filtration, rinsed with excess deionized water, and driedin the vacuum oven at 40° C. overnight. The DF of PSf-g-CH₂Cl determinedthe final weight percent of PEG in PSf-g-PEG. For instance, PSf-g-CH₂Clwith a DF of 0.55 resulted in PSf-g-PEG with 40 wt % PEG. Similarly, DFsof 0.5, 0.4, and 0.23 resulted in PEG loadings of 38 wt. %, 33 wt. %,and 22 wt. %, respectively.

Example 21 Immobilization of Carbonic Anhydrase UsingPolysulfone-Graft-poly(ethylene glycol)

Solvent Evaporation Method. Dry polysulfone-graft-poly(ethylene glycol)(PSf-g-PEG; 0.25 g) and 3.5 mL of dichloromethane were added to a cappedglass vial, and the solution was stirred with a Teflon stir bar todissolve PSf-g-PEG. To a 50 mL beaker, about 6 g of porous supportmaterial and 25 mg of BCA II (Sigma Aldrich; unpurified) or 10 mg of HCAIV (St. Louis University) were added. Once homogenized, the PSf-g-PEGsolution was added to the beaker and stirred to coat the porous supportmaterial. The contents of the beaker were stirred continuously until allof the solvent had evaporated. The coated porous support material wasthen transferred to the vacuum oven at room temperature for 15 minutesto evaporate any residual dichloromethane before transferring to 0.2MNaHCO₃ for storage.

Solvent Exchange Method. Dry polysulfone-graft-poly(ethylene glycol)(PSf-g-PEG; 0.33 g) and 1.5 mL of N-methylpyrrolidone (or a comparablewater-miscible solvent that also dissolves this polymer such as1,4-dioxane) were added to a capped glass vial, and the solution wasstirred with a Teflon stir bar to dissolve PSf-g-PEG. Once homogenized,25 mg of BCA II (Sigma Aldrich; unpurified) or 10 mg of HCA IV (St.Louis University) was added and mixed thoroughly. The solution was thenadded to 250 mL of deionized water dropwise via a transfer pipette toform polymer beads. After 1 hour, the beads were transferred to 0.2MNaHCO₃ for storage.

Example 22 Immobilized BCA II

Unpurified BCA II immobilized in PSf-g-PEG (33 wt % PEG; 550 Da PEG)maintained about 50% of its initial activity after 33 hours (2 nights)at 70° C. A third night (16 additional hours) at 70° C. resulted in aloss of the remaining activity. In contrast, unpurified BCA II free insolution lost its activity after one hour at 70° C. The thermalstability of this immobilized sample compared to free enzyme issummarized in FIG. 23.

The longest lifetime study to date is with unpurified BCA II immobilizedin PSf-g-PEG (22 wt % PEG; 550 Da PEG) on lava rocks, shown in FIG. 24,and unpurified BCA II immobilized in PSf-g-PEG (38 wt % PEG; 550 Da PEG)on lava rocks, shown in FIG. 25. These immobilized samples stilldemonstrate activity after 60 days and 56 days, respectively. Thelifetime of unpurified BCA II free in solution has not been determined.

Example 23 Immobilized HCA IV

PSf-g-PEG was also used to immobilize HCA IV. The greatest thermalstability was found for HCA IV immobilized in PSf-g-PEG (40 wt % PEG;550 Da PEG) where about 100% of its initial activity was maintainedafter 113 hours (7 nights) at 70° C. An eighth night (16 additionalhours) at 70° C., however, resulted in a loss of the remaining activity.The thermal stability of this immobilized sample was compared to freeenzyme and is summarized in FIG. 26. As mentioned previously, thedetermination of the thermal stability of free HCA IV in solution waslimited by the formation of large aggregates after extended heatingtimes. Lifetime studies continue for HCA IV immobilized in PSf-g-PEGsamples. For instance, HCA IV immobilized in PSf-g-PEG (40 wt % PEG; 550Da PEG) demonstrated significant activity after 22 days, as seen in FIG.27.

Example 24 Poly(ethylene glycol) Grafting onto Poly(methylhydrosiloxane)

Poly(methyl hydrosiloxane) (PMHS, MWavg=2250 g/mol; 30 mL) andallyloxy(polyethylene glycol) monomethyl ether (PEG, MWavg=500 g/mol; 27mL) were added to a 500 mL 2-neck round bottom flask equipped withteflon magnetic stir bar. Dry toluene (150 mL, dried over molecularsieves) was then added with stirring to homogenize the solution. Theflask was equipped with a condenser and thermostat and placed in aheating jacket. The flask and condenser was purged with nitrogen whileheating to 80° C. with stirring. Then 0.8 mL of 1 mM chloroplatinic acid(H₂PtCl₆) solution in 2-propanol was injected via a gas-tight syringe.After an additional 5 minutes of nitrogen purging, the purging andventing needles were removed. The reaction mixture was slowly heated to108° C. while stirring and periodically venting the reaction mixture torelieve pressure buildup. The reaction proceeded at 108° C. undernitrogen with stirring for 3 days. The reaction was stopped by coolingto room temperature and then stirring over activated charcoal to removeplatinum catalyst. After 30 minutes, the charcoal was removed viafiltration. About 55 mL of PMHS grafted with PEG (PMHS-g-PEG) wascollected by removing the toluene under reduced pressure. The averagePEG grafting density of this polymer was determined to be 4.8 by ¹H NMR,meaning that it has an average of 4.8 PEG chains per PMHS chain. Thisgrafting density corresponds to a PEG loading of about 52 wt. %. The PEGloading in the PMHS-g-PEG polymers was adjusted by varying the amountsof PEG relative to PMHS used during the reaction. Additional toluene maybe required to homogenize the reaction mixture.

Example 25 Immobilization of CA Using poly(methylhydrosiloxane)-graft-poly(ethylene glycol)

Poly(methyl hydrosiloxane)-graft-poly(ethylene glycol) (1 mL;PMHS-g-PEG; ˜52 wt. % PEG), 1 mL of disilanol terminatedpoly(dimethylsiloxane) (PDMS-(OH)₂, MW_(avg)=4200 g/mol), and 0.15 mL ofsilanol-trimethylsilyl modified Q resin (50 wt. % solution indecamethylcyclopentasiloxane solvent) were mixed together in a glassvial. To this mixture, 50 mg of BCA II (Sigma Aldrich; unpurified) or 30mg HCA IV (St. Louis University) was added and thoroughly mixed.Dibutyldilauryltin catalyst (70 μL) for crosslinking via adehydrogenative coupling mechanism was then mixed in. The solution wastransferred to an acrylic mold of cylinders (¼ inch deep and ⅛ inch indiameter) via a transfer pipette. The mold was placed in therefrigerator at 4° C. for several hours to allow the mixture tocross-link. Once solidified, the polymer pellets (overall ˜25 wt. % PEG)were removed from the mold and stirred in deionized water toequilibrate. The PEG content of the enzyme-containing polysiloxanepieces can be varied by using a PMHS-g-PEG polymer with a different PEGwt. %. As the PEG content increased, some PMHS or hydride Q resin wasadded to improve the cross-link density (10-50 vol. % of the PMHS-g-PEGused). The amount of silanol-trimethylsilyl modified Q resin can also beadjusted to change the cross-link density, ranging from 10-50 vol. % ofthe PMHS-g-PEG used.

A lifetime study was performed of unpurified BCA II immobilized inPMHS-g-PEG (50 wt. % PEG) that was crosslinked using a tin catalyst anddisilanol-terminated PDMS (MW_(avg)=2750 g/mol). The amount of PDMSadded was such that the overall PEG content in the immobilization matrixwas 20 wt. %. A 5-day simple moving average of the % remaining activityin the sample showed the average remaining activity holding fairlysteady around 60% through day 82 of its lifetime.

A study of the thermal stability of unpurified BCA II immobilized inPMHS-g-PEG (50 wt. % PEG) that was crosslinked using a tin catalyst anddi-silanol PDMS (MW_(avg)=2750 g/mol) was performed. The amount of PDMSadded was such that the overall PEG content in the immobilization matrixwas 20 wt. %. The enzyme maintained about 80% of its activity after 32hours at 70° C., whereas BCA II free in solution lost all activity afteronly 1 hour at 70° C.

When introducing elements of the present invention or the preferredembodiments(s) thereof, the articles “a”, “an”, “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising”, “including” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

In view of the above, it will be seen that the several objects of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above compositions and useswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

1-94. (canceled)
 95. A process for removing CO₂ from a CO₂-containinggas, the process comprising contacting an aqueous liquid with aCO₂-containing gas to promote diffusion of the CO₂ into the aqueousliquid; and contacting the CO₂ in the aqueous liquid with immobilizedcarbonic anhydrase entrapped in a polymeric immobilization material tocatalyze hydration of the CO₂ and form a treated liquid containinghydrogen ions and bicarbonate ions; wherein the polymeric immobilizationmaterial either (i) stabilizes the carbonic anhydrase or (ii) comprisesa micellar or inverted micellar material.
 96. The process of claim 95wherein the aqueous liquid and CO₂-containing gas are contacted in aco-current configuration.
 97. The process of claim 96 wherein thepolymeric immobilization material comprises a micellar or invertedmicellar material.
 98. The process of claim 95 wherein the process isperformed in a reaction vessel which comprises a bottom portionincluding a gas inlet and a liquid outlet, a top portion including aliquid inlet and a gas outlet, and a middle portion containing aplurality of particles comprising an immobilized carbonic anhydrase orcoated with immobilized carbonic anhydrase entrapped in a polymericimmobilization material; the process comprising contacting an aqueousliquid which enters the liquid inlet and flows downward in the reactionvessel with a CO₂-containing gas which enters the gas inlet and flowsupward in the reaction vessel to promote diffusion of the CO₂ into theaqueous liquid and catalyze hydration of the CO₂ in the aqueous liquidin the presence of the immobilized carbonic anhydrase to form a treatedliquid containing hydrogen ions and bicarbonate ions and a treated gas;evacuating the treated liquid from the liquid outlet and evacuating thetreated gas from the gas outlet.
 99. The process of claim 95 furthercomprising contacting the treated liquid with particles coated withimmobilized carbonic anhydrase entrapped in a polymeric immobilizationmaterial, wherein the carbonic anhydrase catalyzes conversion of thehydrogen ions and the bicarbonate ions into concentrated CO₂ and water.100. The process of claim 95 wherein the stabilized carbonic anhydraseretains at least about 15% of its initial catalytic activity for atleast about 5 days when continuously catalyzing a chemicaltransformation at a temperature from about 30° C. to about 100° C. 101.The process of claim 98 wherein the CO₂-containing gas enters the gasinlet in the form of microbubbles.
 102. The process of claim 95 whereinthe aqueous liquid comprises a base.
 103. The process of claim 102wherein the base is a metal hydroxide, a quaternary ammonium hydroxide,a metal carbonate, a conjugate base of a weak acid, a quaternaryammonium carbonate, a quaternary ammonium alkoxide, a metal amide, ametal alkyl, a metal alkoxide, metal silanoate, an amine, analkanolamine, or a combination thereof.
 104. The process of claim 95wherein the immobilization material entraps the carbonic anhydrase, theimmobilization material being permeable to a compound smaller than thecarbonic anhydrase and having the structure of either Formulae 5, 6, 7,or 8:

wherein R₂₁ and R₂₂ are independently hydrogen, alkyl, or substitutedalkyl, provided that the average number of alkyl or substituted alkylgroups per repeat unit is at least 0.1; R₂₃ and R₂₄ are independentlyhydrogen, alkyl, or substituted alkyl, provided that the average numberof alkyl or substituted alkyl groups per repeat unit is at least 0.1;R₂₅ is hydrogen, alkyl, or substituted alkyl, provided that the averagenumber of alkyl or substituted alkyl groups per repeat unit is at least0.1; R₃₂ and R₃₃ are independently hydrogen, alkyl, aryl, or substitutedalkyl, provided that the average number of alkyl hydrogen atoms perrepeat unit is at least 0.1 and m, n, o, and p are integers of at least10.
 105. The process of claim 104 wherein the immobilization materialhas a structure of Formula
 8. 106. The process of claim 105 wherein R₃₂and R₃₃ are independently hydrogen, alkyl, aryl, -(substitutedalkylene)-acid or a salt thereof, -(substituted alkylene)-base or a saltthereof, —(CH₂)_(q)O—(CH₂—CH₂—O)_(z)—R_(t),—CH₂—O—(CH₂(CH₃)—CH₂—O)_(z)—R_(t), or a combination thereof, wherein zis an integer corresponding to a weight average molecular weight ofabout 150 Da to about 8000 Da, R_(t) is hydrogen, alkyl, substitutedalkyl, aryl, or substituted aryl, q is an integer of 2, 3, or
 4. 107.The process of claim 106 wherein the acid group comprises a carboxylic,a phosphonic, a phosphoric, a sulfonic, a sulfuric, a sulfamate, a saltthereof, or a combination thereof.
 108. The process of claim 106 whereinthe base comprises a tertiary amine, a quaternary amine, a nitrogenheterocycle, a salt thereof, or a combination thereof.
 109. The processof claim 105 wherein R₃₂ and R₃₃ are independently hydrogen, alkyl,aryl, —(CH₂)₃—O—((CH₂)₂₋₀)_(z)—CH₃, —(CH₂)₂—C(O)—O—(CH₂)₂-imidazolium,or —(CH₂)₃—O—CH₂—CH(OH)—N(CH₃)—(CH₂)₂—SO₃Na and z is an integercorresponding to a weight average molecular weight of about 150 Da toabout 8000 Da.
 110. A system for removing CO₂ from a CO₂-containing gascomprising a reaction vessel having a bottom portion containing a gasinlet and a liquid outlet, a top portion containing a liquid inlet and agas outlet, and a middle portion containing a plurality of particlescomprising an immobilized carbonic anhydrase or coated with carbonicanhydrase entrapped in a polymeric immobilization material, the carbonicanhydrase being capable of catalyzing hydration of CO₂ into hydrogenions and bicarbonate ions, wherein the polymeric immobilization materialeither (i) stabilizes the carbonic anhydrase or (ii) comprises amicellar or inverted micellar material.
 111. The system for removing CO₂from a CO₂-containing gas of claim 110 further comprising a secondreaction vessel, the second reaction vessel containing particles coatedwith carbonic anhydrase entrapped in a polymeric immobilization materialwherein the carbonic anhydrase is capable of catalyzing conversion ofthe hydrogen ions and the bicarbonate ions into concentrated CO₂ andwater.
 112. The system of claim 111 wherein the second reaction vesselcontains an immobilization material comprising a micellar or invertedmicellar material.
 113. An enzyme immobilized by entrapment in polymericimmobilization material, the immobilization material being permeable toa compound smaller than the enzyme and having the structure of eitherFormulae 5, 6, 7, or 8:

wherein R₂₁ and R₂₂ are independently hydrogen, alkyl, or substitutedalkyl, provided that the average number of alkyl or substituted alkylgroups per repeat unit is at least 0.1; R₂₃ and R₂₄ are independentlyhydrogen, alkyl, or substituted alkyl, provided that the average numberof alkyl or substituted alkyl groups per repeat unit is at least 0.1;R₂₅ is hydrogen, alkyl, or substituted alkyl, provided that the averagenumber of alkyl or substituted alkyl groups per repeat unit is at least0.1; R₃₂ and R₃₃ are independently hydrogen, alkyl, aryl, or substitutedalkyl, provided that the average number of hydrogen atoms per repeatunit is at least 0.1 and m, n, o, and p are integers of at least 10.114. An enzyme immobilized by entrapment in a polymeric immobilizationmaterial, the material being permeable to a compound smaller than theenzyme and the enzyme being modified ionically or covalently by ahydrophilic, hydrophobic, or amphiphilic moiety.